Low-toxic broad-spectrum antibacterial peptides containing central pxxp hinge structure and application thereof

Abstract

The application discloses a group of low-toxicity broad-spectrum antibacterial peptides containing a central PXXP hinge structure and application thereof. The low-toxicity broad-spectrum antibacterial peptides are obtained by using a head design method, using positively charged lysine (Lys, K) and hydrophobic tryptophan (Trp, W), symmetrically arranging the two kinds of amino acids alternately and repeatedly, and introducing a PXXP hinge structure containing proline into the symmetry center of the antibacterial peptide sequence. The structural general formula is (KW) n PXXP(WK) n wherein XX=KK, n=2-4 or XX=WK, n=2-3. In-vitro antibacterial experiments show that the antibacterial peptides have broad-spectrum antibacterial activity; the preferred antibacterial peptides have the advantages of low hemolytic toxicity and high serum stability. Induced drug resistance experiments show that the preferred antibacterial peptides have the characteristics of low drug resistance occurrence. In-vivo acute toxicity experiment results show that, compared with a control drug Polymyxin B, the preferred antibacterial peptides have higher in-vivo safety. Therefore, the antibacterial peptides have good application prospects in the preparation of clinical antibacterial drugs and are expected to become new antibiotic candidate drugs.

Description

Technical Field

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

[0002] Traditional antibiotics are effective in treating infectious diseases due to their strong antibacterial activity, but antibiotic overuse has led to the emergence of various drug-resistant bacteria, and the prevalence of antimicrobial resistance is rising rapidly, posing a significant 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.). Because antimicrobial resistance in multidrug-resistant strains can transfer between strains and spread rapidly worldwide, there is an urgent need for new treatment strategies to overcome the limitations of currently available antimicrobial drugs.

[0003] Antimicrobial peptides (AMPs) are a class of small molecule peptides with broad-spectrum antibacterial properties. They can rapidly kill targets and are part of the body's immune defense system. They have become promising candidates for the development of novel antibiotics and have been widely studied for the treatment of infectious diseases related to multidrug-resistant pathogens (Nature Reviews Drug Discovery. 2020, 19(5):311-332.). The most common structural characteristics of AMPs are cationic and amphiphilic. The positive charge of cationic antimicrobial peptides interacts electrostatically with the negatively charged bacterial surface structure and adsorbs them together. The hydrophilic region of the amphiphilic structure of the peptide can insert into the hydrophobic core of the bacterial cell membrane, disrupting the integrity of the bacterial cell membrane and leading to 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 against multidrug-resistant bacteria.

[0004] However, to further apply antimicrobial peptides clinically, it is necessary to overcome the disadvantages of natural AMPs, such as poor stability, high synthesis cost, and high toxicity. Common modification strategies include improving the amphiphilicity of peptides, improving hydrophobicity, and introducing fatty acids or non-natural amino acid substitutions. Wang et al. (Journal of medicinal chemistry, 2018, 61(9):3889-3907.) reported an imperfect amphiphilic palindromic peptide I6 (RRIRIIIRIRR-NH2) that showed strong antimicrobial activity against fungi and bacteria. At the same time, this study showed that most AMPs with incomplete amphiphilic conformations exhibit better broad-spectrum antimicrobial activity. Di et al. (Science advances, 2020, 6(18):eaay6817.) used D-Val to replace L-Val to increase resistance to protease hydrolysis and reduce toxicity to mammalian cells. In addition, secondary structure is also an important parameter affecting the activity of antimicrobial peptides. On the one hand, secondary structure makes an important contribution to maintaining the antimicrobial activity of AMPs. On the other hand, regular and stable secondary structures often lead to increased hemolytic activity and cytotoxicity of AMPs (Frontiers in Microbiology, 2018, 9:2832). Proline (Pro) can serve as an ideal secondary structure disruptor, and inserting Pro into the peptide sequence may improve the selectivity of AMPs. Tripathi et al. (Acta Biomaterialia, 2017, 57:170-86.) attempted to improve cell selectivity by selectively replacing Phe in α-helical peptides with Pro. The results showed that Pro substitution effectively reduced the cytotoxicity of AMPs to mammalian cells without affecting their antibacterial and anti-endotoxin properties. Based on this, the applicant independently designed an amphiphilic antimicrobial peptide formed by alternating 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, aiming to obtain an antimicrobial peptide with high activity, low toxicity, and high stability. Summary of the Invention

[0005] The purpose of this invention is to provide a group of low-toxicity, broad-spectrum antimicrobial peptides containing a central PXXP hinge structure.

[0006] Another object of the present invention is to provide the application of the above-mentioned antimicrobial peptides in the preparation of clinical antimicrobial drugs.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

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

[0009] The low-toxicity, broad-spectrum antimicrobial peptide containing a central PXXP hinge structure provided by this invention has the general structural formula (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, the amino acid sequences of which are as follows:

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

[0012] Or Lys-Trp-Lys-Trp-Lys-Trp-Pro-Lys-Lys-Pro-Trp-Lys-Trp-Lys-Trp-Lys-Trp-Lys-NH2, labeled as (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 as (KW)2PWK;

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

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

[0017] This invention relates to a low-toxicity, broad-spectrum antimicrobial peptide containing a central PXXP hinge structure, synthesized using Rink-MBHA Resin as a raw material and employing the classic Fmoc solid-phase peptide synthesis method. During peptide synthesis, HOBt / HBTU is used as a condensing agent for amino acid coupling, and secondary amines are identified using a ninhydrin colorimetric method. The peptides are sequentially coupled according to their sequences to obtain peptides linked to MBHA resin. The peptides are then cleaved from the MBHA resin and purified by HPLC.

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

[0019] 1. In vitro antibacterial test

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

[0021] Table 1. Minimum inhibitory concentrations of the antimicrobial peptides of this invention against common standard strains.

[0022]

[0023] Table 1 shows that the antimicrobial peptides of this invention have strong inhibitory effects on both Gram-positive and Gram-negative bacteria, exhibiting broad-spectrum antimicrobial activity. The effect against Gram-negative bacteria is superior to Vancomycin; the effect against Gram-positive bacteria is similar to Polymyxin B.

[0024] 2. Hemolytic activity test

[0025] To investigate the toxicity of the antimicrobial peptide synthesized in this invention to normal mammalian cells, the hemolysis of the antimicrobial peptide after co-incubation with mouse erythrocytes for 1 hour was tested. The results are shown below. Figure 6 .

[0026] Figure 6 The results showed that peptides containing the PKKP hinge structure did not exhibit hemolytic activity, and even at a concentration of 256 μM, the hemolytic activity remained close to 0, indicating good safety. Peptides containing the PWKP hinge structure showed significant hemolytic activity when the (KW) unit was repeated 4 times. However, when the (KW) unit was repeated 2 and 3 times, the hemolytic activity at a concentration of 256 μM was also less than 10%, indicating good safety. The hemolytic activity results demonstrate 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] 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 with mouse serum at different time points during co-incubation at 37°C. The serum half-life was determined, and the results are shown below. Figure 7 .

[0029] Figure 7 The results showed that the serum half-life of (KW)3PK was 18.32 h, and the serum half-life of (KW)3PWK was 15.74 h. These results indicate that the antimicrobial peptides containing a central PXXP hinge structure synthesized in this invention exhibit good stability in mouse serum and are not easily degraded by serum enzymes, thus losing their antimicrobial activity.

[0030] 4. Induction of drug resistance experiment

[0031] A major problem with the clinical application of antibiotics is their tendency to induce bacterial resistance. Antimicrobial peptides, due to their membrane-dissolving mechanism, are difficult for bacteria to develop resistance to. Therefore, to investigate whether the antimicrobial peptides of this invention possess the characteristic of not easily inducing resistance, the resistance-inducing effect of antimicrobial peptides (KW)3PK and (KW)3PWK on Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 was determined after 20 days of continuous treatment, with the antibiotics Gentamicin and Polymyxin B as controls. Results are shown below. Figure 8 and Figure 9 .

[0032] Generally speaking, fluctuations in MIC within the range of 1-4 times are normal. 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 induce resistance to Staphylococcus aureus ATCC 25923 after 20 days of continuous treatment, while the antibiotic Gentamicin rapidly induced resistance, with the MIC increasing by 32,768 times after 20 days, and Polymyxin B increasing by 8 times after 20 days. Figure 9 The results showed that the antimicrobial peptides (KW)3PK and (KW)3PWK did not induce resistance in *Pseudomonas aeruginosa* ATCC 27853 after 20 days of continuous treatment, while the antibiotic Gentamicin rapidly induced resistance, with the MIC increasing by 32,768 times after 20 days. Polymyxin B showed a 64-fold increase in MIC after 20 days. Notably, a 1:1 mixture of antimicrobial peptides (KW)3PK and (KW)3PWK with the antibiotic Gentamicin also did not induce resistance. These results indicate that the antimicrobial peptides synthesized in this invention are less likely to induce bacterial resistance compared to traditional antibiotics, and can further reduce the development of antibiotic resistance when used in combination with traditional antibiotics.

[0033] 5. Acute toxicity test in mice

[0034] 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.

[0035] Acute toxicity pre-experiment in mice: Three mice in each group were injected intraperitoneally with antimicrobial peptides (KW)3PK and (KW)3PWK dissolved in physiological saline. Mortality was observed after 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 in mice, and to determine the upper and lower limits of the dose.

[0036] Formal acute toxicity test in mice: The LD50 of (kw) 3 pk was obtained based on the preliminary experimental results. 50 The dosage range is 41.5-80 mg / kg, and the LD50 of (KW)3PWK is... 50 The dosage range is 58.5-100 mg / kg, and the LD50 of the control drug Polymyxin B is... 50 The dosage range was 15-24 mg / kg. Based on the dosage range of (KW)3PK, the common ratio for grouping was calculated to be 1.3884, dividing the mice into three groups with doses ranging from low to high: 41.5 mg / kg, 57.7 mg / kg, and 80 mg / kg. The common ratio for (KW)3PWK was 1.3074, dividing the mice into three groups with doses ranging from low to high: 58.5 mg / kg, 76.6 mg / kg, and 100 mg / kg. The common ratio for the control drug Polymyxin B was 1.2649, dividing the mice into three groups with doses ranging from low to high: 15 mg / kg, 18.9 mg / kg, and 24 mg / kg. Eight mice were administered each dose of each drug to each group. Each group of mice was intraperitoneally injected with the corresponding dose of (KW)3PK, (KW)3PWK, and Polymyxin B. Mortality was observed for 7 days, and the LD50 was recorded and calculated. 50 The results are shown in Table 2. Figure 10 , Figure 11 and Figure 12 .

[0037] Table 2 LD50 of antimicrobial peptides and Polymyxin B in BALB / c mice 50

[0038]

[0039] Figure 10 The results showed that after intraperitoneal injection of 80 mg / kg (KW) 3PK into mice, all 8 mice died within 2 days, with a mortality rate of 100%. After intraperitoneal injection of 57.7 mg / kg (KW) 3PK into mice, 6 out of 8 mice died within 7 days, with a mortality rate of 75%. After intraperitoneal injection of 41.5 mg / kg (KW) 3PK into mice, all mice survived within 7 days. Figure 11The results showed that after intraperitoneal injection of 100 mg / kg of (KW)3PWK into 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 into mice, 3 out of 8 mice died within 7 days, with a mortality rate of 37.5%. After intraperitoneal injection of 58.5 mg / kg of (KW)3PWK into mice, all mice survived within 7 days. Figure 12 The results showed that after intraperitoneal injection of 24 mg / kg of the control drug Polymyxin B into mice, all 8 mice died within 1 hour, with a mortality rate of 100%. After intraperitoneal injection of 18.9 mg / kg of the control drug Polymyxin B into mice, 2 out of 8 mice died within 7 days, with a mortality rate of 25%. After intraperitoneal injection of 15 mg / kg of the control drug Polymyxin B into mice, all mice survived within 7 days. Table 2 shows the calculated LD50 of the antimicrobial peptides (KW)3PK, (KW)3PWK, and the control drug Polymyxin B in BALB / c mice during the acute toxicity test. 50 The LD value of the antimicrobial peptide (KW) 3PK of this invention 50 The LD50 value of the antimicrobial peptide (KW) 3PWK of this invention is approximately 2.65 times that of the control drug Polymyxin B. 50 The value was approximately 3.95 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.

[0040] In summary, this invention employs a de novo design approach, utilizing positively charged lysine (Lys, K) and hydrophobic tryptophan (Trp, W). By alternating and symmetrically arranging these two amino acids and introducing a proline-containing PXXP hinge structure at the center of symmetry of the antimicrobial peptide sequence, a low-toxicity, broad-spectrum antimicrobial peptide is obtained. In vitro antibacterial experiments demonstrate that the antimicrobial peptides of this invention possess broad-spectrum antimicrobial activity. Preferred peptides (KW)3PK and (KW)3PWK exhibit advantages such as low hemolytic toxicity, high serum stability, and low incidence of drug resistance. In vivo acute toxicity experiments show that the antimicrobial peptides (KW)3PK and (KW)3PWK of this invention have higher in vivo safety compared to the control drug Polymyxin B. Therefore, they have excellent application prospects in the preparation of clinical antimicrobial drugs and are expected to become novel antibiotic candidates. Attached Figure Description

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

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

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

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

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

[0046] Figure 6 The results show the hemolytic activity of the antimicrobial peptide of this invention on mouse erythrocytes after incubation for 1 hour.

[0047] Figure 7 The results show the serum stability of the antimicrobial peptides of this invention after co-incubation with mouse serum.

[0048] Figure 8 This invention relates to the antimicrobial peptide of the present invention, and the results of inducing drug resistance after the antimicrobial peptide of the present invention is mixed with the antibiotic Gentamicin and continuously treated with Staphylococcus aureus ATCC 25923 for 20 days.

[0049] Figure 9 The present invention relates to the antimicrobial peptide, and the results of inducing drug resistance after the antimicrobial peptide of the present invention is mixed with the antibiotic Gentamicin and continuously treated with Pseudomonas aeruginosa ATCC 27853 for 20 days.

[0050] Figure 10 This is a diagram showing the results of an in vivo acute toxicity test of the antimicrobial peptide (KW) 3PK of this invention on BALB / c mice;

[0051] Figure 11 The figure shows the results of the in vivo acute toxicity test of the antimicrobial peptide (KW) 3PWK of the present invention on BALB / c mice;

[0052] Figure 12 The figure shows the results of an in vivo acute toxicity test of the control drug Polymyxin B on BALB / C mice. Detailed Implementation

[0053] The synthesis of the low-toxicity broad-spectrum antimicrobial peptide containing a central PXXP hinge structure of the present invention will be further illustrated below through specific embodiments.

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

[0055] (1) Resin activation

[0056] Weigh 0.42 g of MBHA resin (0.48 mmol / g), place it at the bottom of the solid phase synthesizer, add DCM to swell for 30 min, dry under vacuum, wash 3 times with DMF, dry under vacuum again, and identify the resin using the ninhydrin colorimetric method. If the resin is colorless, it is ready for normal use.

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

[0058] The swollen resin was washed three times with a DMF solution containing 20% ​​piperidine, dried, and then washed three more times with DMF, dried again, and identified by ninhydrin colorimetric method. A deep blue-purple color indicates that the Fmoc protecting group has been removed. Three times the excess of Leu, HOBt, HBTU, and six times the 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 ninhydrin colorimetric method. A colorless and transparent resin indicates that Lys condensation was successful, yielding Fmoc-Lys-MBHA.

[0059] Following the above method, Lys, Trp, Lys, Trp, Pro, Lys, Lys, Pro, Trp, Lys, Trp, Lys, Trp, Lys are condensed sequentially to obtain Fmoc-Lys-Trp-Lys-Trp-Pro-Lys-Lys-Pro-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 three times with a DMF solution containing 20% ​​piperidine, dried under vacuum, and washed three times with DMF, dried under vacuum again. The resin was identified by the ninhydrin colorimetric method; if the resin turned deep blue-purple, it indicated that the Fmoc protecting group had been removed. The resin was washed three times alternately with DCM and methanol, and then vacuum-dried for 1 hour. After the resin was dried into dry granules, 10 mL of cleavage reagent (TFA:Tris:water = 9.5:0.25:0.25 (v:v:v)) was added and reacted for 3 hours with slow stirring every 20 minutes. After the reaction time was reached, the cleavage reagent was collected, extracted with diethyl ether, and then freeze-dried.

[0062] (4) Peptide purification

[0063] The lyophilized peptide was purified by RP-HPLC. The RP-HPLC purification conditions were: mobile phase A: 0.1% TFA / acetonitrile, mobile phase B: 0.1% TFA / water, using linear gradient elution. The target peak eluent was collected, lyophilized, and the antimicrobial peptide (KW) 2PK was obtained. Its mass spectrum is shown below. Figure 1 As shown.

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

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

[0066] (1) Resin activation

[0067] Same as Example 1.

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

[0069] Following the method described in Example 1, Lys, Trp, Lys, Trp, Lys, Trp, Pro, Lys, Lys, Pro, Trp, Lys, 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] Same as in Example 1, (KW)3PK was obtained, and its mass spectrum is as follows. Figure 2 As shown.

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

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

[0077] (1) Resin activation

[0078] Same as Example 1.

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

[0080] Following the method described in Example 1, 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 in Example 1, (KW)4PK was obtained, and its mass spectrum is as follows. Figure 3 As shown.

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

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

[0088] (1) Resin activation

[0089] Same as Example 1.

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

[0091] The same method as in Example 1 is used to sequentially condense Lys, Trp, Lys, Trp, Pro, Lys, Trp, Pro, Trp, Lys, Trp, Lys 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 As shown.

[0096] The theoretical molecular weight calculation result 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) Resin activation

[0099] Same as Example 1.

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

[0101] Following the method described in Example 1, Lys, Trp, Lys, Trp, Lys, Trp, Pro, Lys, Trp, Pro, Trp, Lys, Trp, Lys, Trp, Lys, Trp, Lys are sequentially condensed 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 in Example 1, (KW)3PWK was obtained, and its mass spectrum is as follows. Figure 5 As shown.

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

Claims

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

2. The set of low toxic broad spectrum antibacterial peptides with central PXXP hinge structure according to claim 1, wherein, The low-toxic broad-spectrum antibacterial peptide is (KW) 3PK or (KW) 3PWK.

3. The set of low toxic broad spectrum antibacterial peptides with central PXXP hinge structure as claimed in claim 2, wherein, The low-toxic broad-spectrum antibacterial peptide is (KW) 3PK.

4. Use of the low toxic broad-spectrum antibacterial peptide containing the central PXXP hinge structure according to any one of claims 1-3 for the preparation of a clinical antibacterial drug, characterized in that, The bacteria inhibited by the low-toxic broad-spectrum antibacterial peptide are gram-positive bacteria or gram-negative bacteria; the gram-positive bacteria are Staphylococcus aureus or Bacillus subtilis, and the gram-negative bacteria are Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa or Acinetobacter baumannii.

5. The use of the low toxic broad-spectrum antibacterial peptide containing the central PXXP hinge structure according to claim 4 in combination with the antibiotic Gentamicin for the preparation of clinical antibacterial drugs, characterized in that, The antibacterial peptide is (KW) 3PK or (KW) 3PWK.

6. The use of the low toxic broad-spectrum antibacterial peptide containing the central PXXP hinge structure according to claim 5 in combination with the antibiotic Gentamicin for the preparation of clinical antibacterial drugs, characterized in that, The mixing ratio of the antibacterial peptide (KW) 3PK or (KW) 3PWK and the antibiotic Gentamicin is 1:1.

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