CN120699105B

CN120699105B - A deuterated antimicrobial peptide, its composition and application

Info

Publication number: CN120699105B
Application number: CN202511157997.6A
Authority: CN
Inventors: 牛淼淼
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2025-08-19
Filing date: 2025-08-19
Publication date: 2025-11-21
Anticipated expiration: 2045-08-19
Also published as: CN120699105A

Abstract

The invention discloses deuterated antibacterial peptide, and a composition and application thereof, and belongs to the field of biological medicines. According to the invention, the deuterated 2-aminobutyric acid is introduced to form a ring structure, and deuterated modification is combined, so that the affinity of the antibacterial peptide to a bacterial membrane is greatly improved compared with that of a traditional medicament (polymyxin B, D), the selectivity to a mammalian membrane is more than 884 times of that of a control substance, the minimum antibacterial concentration to gram-negative bacteria in vitro is as low as 0.015 mug/mL, more colistin B is improved by 67 times, 33-67 times than that of a D50 control peptide, and about 17 times than that of a non-deuterated antibacterial peptide 5, the extremely low concentration efficient antibacterial effect is realized, the toxicity to human kidney cells is extremely low, and the biological safety is better. In various infection models, the therapeutic effect of the composition is obviously superior to that of the three controls, the composition can be used for preparing anti-infective drugs, provides a new scheme of high efficiency and low toxicity for clinical anti-infective treatment, and is suitable for preventing and treating various infections.

Description

Deuterated antibacterial peptide, and composition and application thereof

Technical Field

The invention relates to a polypeptide and application thereof, in particular to a deuterated antibacterial peptide, and a composition and application thereof.

Background

The current bacterial drug resistance problem is increasingly serious, particularly gram negative bacteria, such as escherichia coli, klebsiella pneumoniae, acinetobacter baumannii and pseudomonas aeruginosa, and the bacteria can build a complex drug resistance network by means of biological mechanisms such as horizontal gene transfer, efflux pump mechanism and the like, thereby bringing great technical challenges to clinical treatment links. Wherein, the carbapenem drug-resistant klebsiella pneumoniae has a high mortality rate and forms a remarkable threat to public health safety.

Polymyxin, a drug for the treatment of drug-resistant gram-negative bacterial infections, has the mechanism of action of being able to bind specifically to the outer membrane lipopolysaccharide lipid a of bacteria. However, in practical application, the drug-resistant strain has obvious technical defects that on one hand, the drug-resistant strain can carry out phosphoethanolamine modification on lipopolysaccharide, the modification effect can lead to the great reduction of the affinity between a drug and bacteria, and simultaneously an efflux system is activated, so that drug resistance phenomenon appears rapidly, the technical problem of interaction between a bacterial drug resistance mechanism and a drug action mechanism is related, the effectiveness of polymyxin in drug-resistant bacteria treatment is difficult to be sustained, on the other hand, in the process of using polymyxin treatment, a patient is easy to suffer irreversible tubular injury, and adverse reactions such as neurotoxicity and ototoxicity are accompanied, the therapeutic index is low, the requirements of clinical treatment on the safety and the effectiveness of the drug are difficult to meet, and the limitation of the polymyxin is highlighted on the technical engagement of drug development and clinical application.

The antimicrobial peptide is used as a natural amphiphilic molecule, the action mechanism of the antimicrobial peptide is to play an antibacterial role by destroying bacterial cell membranes, the killing efficiency of the antimicrobial peptide on resistant bacteria is higher than that of traditional antibiotics in technical characteristics, and the antimicrobial peptide can still keep good activity in a high-temperature environment. However, some antibacterial peptides disclosed in the prior art, such as the optimal antibacterial peptide D50 reported in patent CN106232617A, have the problems of insufficient activity and higher cytotoxicity to gram-negative bacteria in the technical effect aspect of practical application, which limit the further application of the antibacterial peptide in the clinical transformation technical process of the antibacterial peptide, so that the development of a novel antibacterial peptide with high-efficiency antibacterial activity, low toxicity and high biological safety is of great significance in breaking the clinical treatment dilemma caused by drug-resistant gram-negative bacteria and meeting the requirements of clinical treatment in the technical and application aspects from the technical targets of clinical treatment requirements and antibacterial drug research and development.

Disclosure of Invention

The invention aims to provide deuterated antibacterial peptide and a pharmaceutical composition thereof, so as to provide more choices for resisting microbial infection, in particular to resisting gram-negative bacterial infection.

The specific structure of the antibacterial peptide is any one of the following (I) - (IV):

(1) As shown in formula (I):

;

(2) As shown in formula (II):

;

(3) As shown in formula (III):

;

(4) As shown in formula (IV):

。

the pharmaceutical composition contains the antibacterial peptide or pharmaceutically acceptable salt, ester, solvate, hydrate or prodrug thereof as an active ingredient, and pharmaceutically acceptable auxiliary materials are added or not added.

The pharmaceutical composition comprises one or more of excipient, diluent, lubricant, glidant, wetting agent, emulsifier and pH buffer substance.

The dosage forms of the pharmaceutical composition comprise tablets, capsules, granules, oral liquid, syrup, powder, chewable tablets, effervescent tablets, sustained-release tablets, micropills, injection, powder injection, infusion solution, suspension, ointment, cream, gel, spray, eye drops, ear drops, nose drops, patches, lotions, suppositories, atomized solutions, film coating agents, implants, orally disintegrating tablets, oral instant films, sponges and capsules.

The application of the antibacterial peptide or the pharmaceutical composition in preparing medicines for preventing and/or controlling microbial infection.

For said use, said microbial infection is a bacterial infection.

The use, the bacterium is a gram-negative bacterium.

The application is that the gram negative bacteria are one or more of escherichia coli, klebsiella pneumoniae, acinetobacter baumannii and pseudomonas aeruginosa.

The application is that the microorganism infection is one or more of respiratory system infection, urinary system infection, motor system infection, skin soft tissue infection, systemic infection type, circulatory system infection, digestive system infection, nervous system infection, endocrine system infection and reproductive system infection.

The use of the microbial infection comprises urinary tract infection, pneumonia, burn infection, peritonitis, cholecystitis, pyelonephritis, septicemia, hospital-acquired pneumonia, wound infection, neonatal meningitis, necrotizing fasciitis, liver abscess, osteomyelitis, suppurative arthritis, endocarditis, keratitis, otitis media, sinusitis, cellulitis, ventilator associated pneumonia, melioidosis, chancroid, legionella disease, gastritis, peptic ulcer, cholangitis.

Preferably, the "pharmaceutically acceptable excipients" are substances suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation and allergic response), i.e. with a reasonable benefit/risk ratio, and also include various excipients and diluents, which may contain liquids, such as water, saline, glycerol and ethanol, or auxiliary substances, such as lubricants, glidants, wetting agents, emulsifiers, pH buffering substances.

Preferably, the pharmaceutical dosage form comprises injection, oral preparation or external preparation, and the external preparation comprises eye drops or lotion, wherein the dosage range of the antibacterial peptide in the dosage form is 0.001-1000mg/kg of injection, 0.001-1000mg/kg of oral preparation, 1/10000-30% per unit of external preparation, 1/10000-30% per unit of eye drops and 1/100000-20% per unit of lotion.

The core innovation content of the invention is as follows:

1. conformational innovation, breaking through the action mechanism of traditional polypeptide

The novel annular structure design is that the mother nucleus forms an annular conformation different from a polymyxin B linear structure and a D50 control peptide through the introduction of deuterated 2-aminobutyric acid, so that the ultra-high affinity to a bacterial membrane is improved (more colistin B is improved by 140-280 times and is improved by 75-150 times and is improved by 21-43 times compared with D50 and is improved by 21-43 times compared with non-deuterated antibacterial peptide 5), and the novel annular structure design has extremely strong selectivity to a mammal membrane (more than 884 times compared with the polymyxin B and the control peptide D50), and is proved to be capable of accurately combining the bacterial membrane to play an antibacterial effect and avoiding damage to the mammal membrane. The characteristics of targeting bacteria and protecting hosts enable the antibacterial peptide to show more excellent application potential-double breakthrough of affinity and selectivity compared with the traditional polymyxin B and the control peptide D50 in the development of novel antibacterial drugs, and the antibacterial peptide is a core advantage of the antibacterial peptide 1-4 different from the traditional peptides. Furthermore, the unique advantage of deuterated modifications is that antimicrobial peptides 1-4 have about 17-fold improved MIC values for all four bacteria compared to non-deuterated antimicrobial peptide 5.

The value is that the conformation innovation breaks through the drug resistance barrier of the traditional antibiotics from the molecular level, and provides a new paradigm for the development of low-toxicity high-efficiency antibacterial peptides.

2. The antibacterial activity is high-efficiency for eliminating drug-resistant bacteria and has remarkable dose dependency

The in-vitro antibacterial efficacy breaks through that the minimum antibacterial concentration (MIC) of 4 gram-negative bacteria such as escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa and the like is as low as 0.015 mug/mL, more colistin B (MIC=1 mug/mL) is improved by 67 times, 33-67 times as compared with D50 control peptide, about 17 times as compared with non-deuterated antibacterial peptide 5, and the ultra-low concentration high-efficiency antibacterial effect is realized.

The in-vivo efficacy has remarkable advantages that in a rat thigh infection model, the antibacterial peptide has more outstanding in-vivo antibacterial effect compared with polymyxin B, D control peptide and non-deuterated antibacterial peptide 5, shows that the antibacterial peptide has better antibacterial effect than the control medicament under the same dosage, and has definite dose dependence (dose increase and antibacterial effect linear enhancement).

The method has the advantages of solving the difficult problem of high mortality rate and difficult removal of clinical multi-drug resistant bacteria and providing a new treatment scheme for super bacterial infection such as carbapenem drug resistant bacteria.

3. Safety optimization, low toxicity property breaking through bottleneck of traditional antibiotics

The toxicity of the antibacterial peptide to HK-2 human kidney proximal tubule epithelial cells is improved by 87 times, compared with D50 control peptide (IC ₅₀ =5), the safety of the antibacterial peptide to colistin B (IC ₅₀ =1) is improved by about 17 times, compared with non-deuterated antibacterial peptide 5, the antibacterial peptide is improved by about 5.8 times, and the clinical dilemma of high toxicity and low efficiency of polymyxin B and control peptide D50 is avoided.

MST experiments prove that the antibacterial peptide 1-4 can accurately combine with the bacterial membrane to play antibacterial role and avoid damage to the mammalian membrane through ultrahigh affinity (140-280 times for more colistin B, 75-150 times for D50 and 21-43 times for non-deuterated antibacterial peptide 5) and extremely strong selectivity (884 times for more than control polymyxin B and control peptide D50) for the mammalian membrane.

The value of the antibiotic breaks through the limit of narrow treatment window of the traditional antibiotic, and provides safe selection for long-term medication or immunodeficiency patients.

Compared with the prior art, the antibacterial peptide has the advantages that (1) the antibacterial peptide is a novel efficient broad-spectrum antibacterial peptide, and 1-4 of the antibacterial peptide has excellent antibacterial activity on various gram-negative bacteria such as escherichia coli, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa and the like, and in-vitro and in-vivo experiments prove that the antibacterial efficacy of the antibacterial peptide is obviously better than that of polymyxin B, D peptide and non-deuterated antibacterial peptide 5, and the antibacterial peptide has good dose dependence, so that a novel effective means is provided for treating multiple drug-resistant gram-negative bacteria infection; the antibacterial peptide is a low-toxicity and safe antibacterial peptide, has extremely low toxicity to HK-2 cells, has higher safety than colistin B and D50 contrast peptide, is expected to reduce adverse reactions such as nephrotoxicity and the like in the traditional antibiotic treatment process, improves the tolerance and treatment safety of patients, is not easy to induce bacteria to generate drug resistance by destroying the action mechanism of bacterial cell membranes, overcomes the defect of reduced curative effect caused by the drug resistance problem of the traditional antibiotics, provides a new thought and direction for solving the global drug resistance crisis, has the advantages of good biocompatibility, high stability, low synthesis cost and the like, has the advantages of being excellent in treatment effect in various infection models, has great potential of developing novel anti-infective drugs, is expected to be applied to the prevention and treatment of various infections such as respiratory systems, urinary systems, skin soft tissues and the like, brings breakthrough progress for the treatment of clinical multiple drug resistance infection, and is expected to become a safe and green substitute antibiotics, high efficacy of the ideal antimicrobial agent.

Drawings

FIG. 1 is an HPLC chromatogram of antibacterial peptide 1;

FIG. 2 is an HPLC chromatogram of antibacterial peptide 2;

FIG. 3 is an HPLC chromatogram of antibacterial peptide 3;

FIG. 4 is an HPLC plot of antibacterial peptide 4;

FIG. 5 shows the results of an in vitro renal cytotoxicity assay of antimicrobial peptides.

Detailed Description

The technical scheme of the invention is further described below.

The polypeptide compound in the embodiment of the invention, the antibacterial peptide 1 is:

antibacterial peptide 2 is:

Antibacterial peptide 3 is:

Antibacterial peptide 4 is:

Example 1 preparation of antibacterial peptides 1-4

1. Preparation and purification of antibacterial peptide 1

(1) Preparation of 4- ((tert-Butoxycarbonyl) amino) -3- (3-isopropylphenyl) -butanoic acid

A mixture of 10g of (E) -3- (3-isopropylphenyl) acrylic acid, 100mL of methanol and 4mL of concentrated sulfuric acid was stirred at room temperature for 20 hours. The mixture was evaporated to dryness and the residue partitioned between Dichloromethane (DCM) and water. The aqueous phase was separated and extracted with dichloromethane. The organic extracts were combined, dried over magnesium sulfate, filtered and evaporated to dryness to give 10.19g of a white solid.

32ML of a mixture of nitromethane and 8.5mL of 1, 8-diazabicyclo [5.4.0] undec-7-ene (DBU) was added and stirred at room temperature for 20 hours. The mixture was evaporated to dryness and the residue partitioned between 0.5M aqueous hydrochloric acid and diethyl ether. The aqueous phase was separated and extracted with diethyl ether. The organic extracts were combined, washed with brine, dried over magnesium sulfate, filtered and evaporated to dryness. The residue was purified on silica gel eluting with hexane and ethyl acetate (0-100%). Evaporation to dryness gave 9.93g of yellow oil.

To a stirred solution of yellow oil in 90mL of acetic acid at 0℃was added 20.1g of zinc powder. The mixture was warmed to room temperature and stirred for 19 hours. The mixture was evaporated to dryness and the residue partitioned between NaHCO ₃ solution and ethyl acetate. The mixture was then filtered through celite, separating the aqueous and organic phases. The aqueous phase was extracted with ethyl acetate. The organic extracts were combined, washed with brine, dried over magnesium sulfate, filtered and evaporated to dryness to give 4.80g of an orange oil.

The orange oil obtained in the previous step was dissolved in 100mL of dichloromethane and treated with 5.28g of di-tert-butyl dicarbonate and the mixture was stirred at room temperature for 18 hours. The mixture was evaporated to dryness and the residue was purified on silica gel eluting with 0-100% ethyl acetate in hexane. Evaporation to dryness gave the desired milky white solid product, 2.59g.

A mixture of 2.59g of a milky white solid product, 546mg of lithium hydroxide, 40mL of 1, 4-dioxane and 40mL of water was stirred at room temperature for 64 hours. The mixture was evaporated to dryness. The residue was dissolved in water, neutralized with 1M HCl solution, and extracted 2 times with ethyl acetate. The organic extracts were combined, washed with brine, dried over magnesium sulfate, filtered and evaporated to dryness to give 2.51g of 4- ((tert-butoxycarbonyl) amino) -3- (3-isopropylphenyl) -butanoic acid as a yellow oil.

After purification of 4- ((t-butoxycarbonyl) amino) -3- (3-isopropylphenyl) butanoic acid by High Performance Liquid Chromatography (HPLC), the fractions were evaporated under reduced pressure at 40 ℃ bath temperature to give the desired enantiomer as the title compound.

High performance liquid chromatograph (Agilent 1200), chromatographic column (Phenomenex Hyperclone C BDS,5 μm,4.6 mm ×150 mm), detection wavelength (210, 254, nm), mobile phase A phase (water/acetonitrile (90/10, volume ratio) containing 0.15% TFA, mobile phase B phase (acetonitrile/water (90/10, volume ratio) containing 0.15% TFA, flow rate (1 mL/min), sample loading (20. Mu.L), elution gradient (0 min,100% mobile phase A), 20min,40% mobile phase A,60% mobile phase B, 21min,100% mobile phase B, 23min,100% mobile phase B, 23.5min,100% mobile phase A, 25min,100% mobile phase A).

(2) The cyclic peptides were prepared by an automated polypeptide synthesizer using standard Fmoc solid-phase polypeptide synthesis. 10g of CTC-Resin with the substitution degree of 0.5+/-0.1 mmol/g is added into a polypeptide reactor, 2g of Fmoc-Thr (tBu) -OH and 10mL of N, N-Diisopropylethylamine (DIEA) are added, the mixture is placed on a shaker to react for 2 hours by gentle shaking, the reaction solution is pumped out and the DMF is washed twice, and after Fmoc protection is removed, the N, N-Dimethylformamide (DMF) is washed twice, dichloromethane (DCM) is washed once and the DMF is washed twice;

(3) Amino acids in the same amount as the above are added sequentially to react, including 2.22g Fmoc-Dab(Boc)-OH,2.22g Fmoc-Dab(Boc)-OH, 1.65g Fmoc-Abu-OH-D3, 1.95g Fmoc-D-Phe-OH, 2.22g Fmoc-Dab(Boc)-OH, 1.72g Fmoc-Dab-OH, 2.15g Fmoc-Dap(Boc)-OH, 2g Fmoc-Thr(tBu)-OH, 2.22g Fmoc-Dab(Boc)-OH, 1.89g 4-[( t-butoxycarbonyl) amino ] -3- (3-isopropylphenyl) butanoic acid. 10 mL of N, N-Diisopropylethylamine (DIEA) was added to each of the added polypeptides, the mixture was placed on a shaker, gently shaken, the reaction time was 2 hours, the reaction solution was withdrawn, and the DMF was washed twice. Fmoc deprotection was performed using 20% piperidine in N, N-dimethylformamide for a deprotection time of 0.5 hours.

(4) After the reaction was completed, DMF was washed twice, once with DCM and twice with DMF. The washed resin was washed twice with methanol, and then, the resin was removed with 200mL of trifluoroethanol/methylene chloride in a volume ratio of 1:2 for 2 hours each time, and the resin was filtered off, and the reaction mixture was collected and dried by spin to give 6.8g of (S) -4-amino-3- (3-isopropylphenyl) butyryl-Thr (tBu) -Dap (Boc) -Dab (Boc) -D-Phe-Abu-D3-Dab (Boc) -Thr (tBu) -OH;

(5) 6.8g of (S) -4-amino-3- (3-isopropylphenyl) butyryl-Thr (tBu) -Dap (Boc) -Dab (Boc) -D-Phe-Abu-D3-Dab (Boc) -Thr (tBu) -OH was dissolved in 20mL of DMF, 0.5g of 1-Hydroxybenzotriazole (HOBT) and 0.5mL of N, N' -Diisopropylcarbodiimide (DIC) were added and stirred at 400rpm at room temperature for 2 hours, the reaction solution was poured into 200mL of ice water, and after stirring with a magnet at 400rpm for 2 hours, the solid was obtained by suction filtration, the cake was rinsed 2 times with 50mL of water and dried with an oil pump to obtain 5.4g of white solid (S) -4-amino-3- (3-isopropylphenyl) -Dap (Boc) -Cyclo (Dab-D-Phe-D3-Dab (Boc) -Ab (Boc) -OH);

(6) 5.4g of the product obtained in the previous step was dissolved in 60mL of trifluoroacetic acid/water/triisopropylsilane (volume ratio 8:1:1) solution, stirred at 15 ℃ and 400rpm for 3 hours, added dropwise to 500mL of glacial diethyl ether solution, centrifuged by precipitation, washed three times with diethyl ether, and dried under reduced pressure to obtain 2.2g of (S) -4-amino-3- (3-isopropylphenyl) butyryl-Thr-Dap-Cyclo (Dab-D-Phe-Abu-D3-Dab-Thr-OH) as a white solid.

(7) The product obtained in the previous step was dissolved in 10ml of 10% acetonitrile-water solution, and after filtration, purified by High Performance Liquid Chromatography (HPLC) to obtain 1.1g of the antibacterial peptide 1 with an HPLC purity of 98.56% (see FIG. 1). The chromatographic conditions were as follows:

High performance liquid chromatograph (Agilent 1200), chromatographic column (Phenomenex Hyperclone BDS C, 5 μm,4.6 mm ×150 mm), detection wavelength (210 nm), mobile phase A phase (water/acetonitrile (90/10, volume ratio) containing 0.15% TFA, mobile phase B phase (acetonitrile/water (90/10, volume ratio) containing 0.15% TFA), flow rate (1 mL/min), elution gradient (0 min,100% mobile phase A, 20min,40% mobile phase A,60% mobile phase B, 21min,0% mobile phase A,100% mobile phase B, 23min,0% mobile phase A,100% mobile phase B, 23.5min,100% mobile phase A, 25min,100% mobile phase A).

2. Preparation and purification of antibacterial peptide 2

4- ((Tert-Butoxycarbonyl) amino) -3- (3-chlorophenyl) -butyric acid was prepared as in step (1) above starting from 10 g (E) -3- (3-chlorophenyl) acrylic acid, 100 mL methanol and 4 mL concentrated sulfuric acid. And (3) sequentially performing nitration, reduction, boc protection, hydrolysis, peptide coupling and deprotection reactions in the steps (2) to (7). Wherein 2.22g Fmoc-Dab(Boc)-OH,2.22g Fmoc-Dab(Boc)-OH, 1.65g Fmoc-Abu-OH-D3, 1.78g Fmoc-D-Leu-OH, 2.22g Fmoc-Dab(Boc)-OH, 1.72g Fmoc-Dab-OH, 2.15g Fmoc-Dap(Boc)-OH, 2g Fmoc-Thr(tBu)-OH, 2.22g Fmoc-Dab(Boc)-OH, 1.89g 4-[( t-butoxycarbonyl) amino ] -3- (3-chlorophenyl) butanoic acid was added to step (3). The crude product was purified by HPLC to give antibacterial peptide 2 with an HPLC purity of 98.32% (see fig. 2).

3. Preparation and purification of antibacterial peptide 3

After preparing 4- ((t-butoxycarbonyl) amino) -3- (3-chlorophenyl) -butyric acid as in step (1) above using 10 g (E) -3- (3-chlorophenyl) acrylic acid, 100 mL methanol and 4 mL concentrated sulfuric acid as starting materials. And (3) performing nitration, reduction, boc protection, hydrolysis, peptide coupling and deprotection reaction in sequence in the steps (2) to (7), and purifying the crude product by HPLC to obtain the antibacterial peptide 3 with the HPLC purity of 98.39% (see figure 3).

4. Preparation and purification of antibacterial peptide 4

4- ((Tert-Butoxycarbonyl) amino) -3- (3, 5-dichlorophenyl) -butyric acid was prepared as in step (1) above starting from 10 g (E) -3- (3, 5-dichlorophenyl) acrylic acid, 100 mL methanol and 4 mL concentrated sulfuric acid. And (3) sequentially performing nitration, reduction, boc protection, hydrolysis, peptide coupling and deprotection reaction in the steps (2) - (7), wherein 2.22g Fmoc-Dab(Boc)-OH,2.22g Fmoc-Dab(Boc)-OH, 1.65g Fmoc-Abu-OH-D3, 1.78g Fmoc-D-Nle-OH, 2.22g Fmoc-Dab(Boc)-OH, 1.72g Fmoc-Dab-OH, 2.15g Fmoc-Dap(Boc)-OH, 2g Fmoc-Thr(tBu)-OH, 2.22g Fmoc-Dab(Boc)-OH, 1.89g 4-[( t-butoxycarbonyl) amino ] -3- (3, 5-dichlorophenyl) butyric acid is added in the step (3). The crude product was purified by HPLC to give antibacterial peptide 4 with HPLC purity 98.12 (see fig. 4).

As can be seen by combining the HPLC chromatograms of FIGS. 1-4, the main peak area ratio of each antibacterial peptide exceeds 98% (98.56% for antibacterial peptide 1, 98.32% for antibacterial peptide 2, 98.39% for antibacterial peptide 3, 98.12% for antibacterial peptide 4), indicating that the prepared antibacterial peptides 1-4 have higher purity. The peptides prepared above were identified and analyzed using Agilent 1200 tandem AB SCIEX API3200, the results of which are shown in figures 1-4 and table 1:

example 2 micro-thermophoresis (MST) determination of antibacterial peptide interactions with lipids

In this study, polymyxin B, D peptide and non-deuterated antibacterial peptide 5 were used as control peptides, prepared and synthesized by Shanghai Jier Biochemical Co., ltd (purity > 96%) and used without further purification.

1, 2-Dimyristoyl phosphatidylglycerol (DMPG, avanti Polar Lipids, product No. 840445) or 1, 2-dimyristoyl phosphatidylcholine (DMPC, avanti Polar Lipids, product No. 850345) is respectively weighed into a flask, chloroform-methanol mixed solution with a volume ratio of 3:1 is added for fully dissolving to prepare clear lipid solution, the lipid solution is dried under nitrogen flow and placed in a vacuum environment overnight to obtain a DMPG and a DMPC lipid film, phosphate buffer with a pH value of 7.4 and a concentration of 10 mmol/L is added for hydration, the final concentration of the DMPG or the DMPC lipid reaches 100 mu mol/L, and after horizontal oscillation for 1h at room temperature, a Sonics Vibra-Cell ultrasonic Cell breaker is used for carrying out 150W power ultrasonic treatment on the system for 0.5 hours to prepare the DMPG or the DMPC liposome. The particle size of the liposomes prepared was determined by dynamic light scattering using Zetasizer Nano ZS instrument from malv instruments limited in the united kingdom. The results showed that the particle sizes of DMPG and DMPC liposomes were 101±8 nm and 380±11 nm, respectively. Dissolving 1-5 or polymyxin B or D50 peptide in PBS with pH=7.4 to obtain 200 nM solution, preparing serial diluted solution with liposome concentration of 100,50,25,12.5,6.25,3.125,1.563,0.781,0.391,0.195,0.098,0.049,0.024,0.012,0.006,0.003 μmol/L, mixing the above-mentioned antibacterial peptide solution and serial diluted solution, injecting into standard glass capillary tube, detecting with NanoTemper Monolith NT.115 molecular interaction instrument, setting MST experimental parameters as LED power (heating solution) 50%, MST power (blue light excitation) 20%, opening and closing time 30 s and 5 s respectively, controlling experimental temperature at 37 deg.C, and performing data analysis by NanoTemper Analysis software to obtain the affinities (K _d, μM) of 1-5, polymyxin B and D50 peptide to DMPG and DMPC, and the results are shown in Table 2 below.

The results are shown in Table 2, and the antibacterial peptides 1-4 prove that the antibacterial peptide can accurately combine with the bacterial membrane to play antibacterial action and avoid damage to the mammalian membrane through the ultrahigh affinity (140-280 times for more colistin B, 75-150 times for D50 and 21-43 times for more non-deuterated antibacterial peptide 5) and the extremely strong selectivity (884 times or more for more than control polymyxin B and control peptide D50) for the bacterial membrane DMPC. The characteristics of targeting bacteria and protecting hosts enable the antibacterial peptide to show more excellent application potential-double breakthrough of affinity and selectivity compared with the traditional polymyxin B, the control peptide D50 and the non-deuterated antibacterial peptide 5 in the development of novel antibacterial drugs, and are the core advantages of the antibacterial peptides 1-4 different from the traditional peptides.

Example 3 antibacterial Activity experiment of antibacterial peptide

According to the standard operating specification of CLSI (clinical laboratory standards institute) M07-A10, minimum Inhibitory Concentrations (MIC) of antimicrobial peptides 1-5 were determined for 4 clinically common gram-negative bacteria including E.coli ATCC25922, klebsiella pneumoniae ATCC43816, pseudomonas aeruginosa ATCC27853, acinetobacter baumannii NCTC13424 using a miniserial double dilution method.

The D50 control peptide was synthesized as a control according to the synthesis method disclosed in CN106232617 a.

(1) Bacterial colonies of fresh escherichia coli ATCC25922, klebsiella pneumoniae ATCC43816, pseudomonas aeruginosa ATCC27853 and acinetobacter baumannii NCTC13424 which are cultured on Mueller-Hinton (MH) agar plates for 18 hours under the aerobic condition of 35 ℃ are respectively taken, bacterial suspension is prepared by adopting sterile physiological saline, and the bacterial suspension is calibrated to 0.5 Maillard turbidity by a turbidimetry method;

(2) Double gradient dilution is carried out on the to-be-detected antibacterial peptide 1-5, D50 control peptide or polymyxin B by using cation adjustment MH broth in a sterile 96-well microplate to form antibacterial solutions with concentration gradients of 0.0075, 0.015, 0.03, 0.06, 0.125, 0.25, 0.5, 1,2, 4, 8, 16, 32 and 64 mug/mL, the final concentration of DMSO is 0.1 percent, and 150 mug of gradient dilution liquid is preset in each well;

(3) Quantitatively inoculating 20 mu L of standardized bacterial suspension to each well to make the final volume of the system be 170 mu L/well, and setting 2 parallel repeated wells for each test concentration;

(4) The microplates were then placed in a 35 ℃ incubator for incubation at rest for 20 hours. The lowest drug concentration that completely inhibited the visible growth of the microorganism was determined by visual observation, with turbidity disappearing as a determination endpoint.

For the compound subjected to five or more independent repeated experiments, the numerical median of each experimental result is taken as a final MIC value, and the concentration units are uniformly expressed by mug/mL.

As a result, as shown in Table 3, in the Minimum Inhibitory Concentration (MIC) measurement against Escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, acinetobacter baumannii, the MIC values of the antimicrobial peptides 1 to 4 were 0.015. Mu.g/mL, and the MIC value of polymyxin B was 1. Mu.g/mL. Experimental data shows that the MIC values of the antibacterial peptides 1-4 are improved by about 67 times for four bacteria compared with polymyxin B, about 33 times for Escherichia coli, klebsiella pneumoniae and Pseudomonas aeruginosa compared with D50 control peptide, about 67 times for Acinetobacter baumannii, and about 17 times for four bacteria compared with non-deuterated antibacterial peptide 5. This shows that the antibacterial peptide can effectively inhibit the growth of gram-negative bacteria at extremely low concentration, and shows in vitro antibacterial efficacy far exceeding that of traditional antibiotics. By virtue of the characteristics, the antibacterial peptide 1-4 is hopeful to reduce the potential toxic and side reaction risks by reducing the dosage in application, constructs a key foundation for developing novel anti-infective agents, has potential worthy of deep excavation in the clinical transformation process, or can bring innovative breakthrough to the existing pattern of anti-infective treatment, and provides a new exploration direction for solving the clinical problems related to bacterial infection.

Example 4 in vitro renal cytotoxicity assay of antibacterial peptides

Cytotoxicity was evaluated using 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2H-tetrazolium bromide (MTT) colorimetry.

(1) HK-2 human kidney proximal tubule epithelial cells CRL-2190 were inoculated in DMEM medium containing 10% fetal bovine serum, cultured to logarithmic growth phase at 37 ℃ under 5% CO ₂, inoculated in 96 well plates at a density of 2.5×103 cells/well, pre-cultured for 24 hours until cells were fully adherent;

(2) Adding 1-5, D50 control peptide or polymyxin B diluted by multiple ratio into the adherent cell hole, and continuously exposing and culturing for 48 hours, wherein the final concentration gradient is 0.0075, 0.015, 0.03, 0.06, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64 and 128 mug/mL;

(3) After the drug-containing culture medium is removed, 110 mu L of newly prepared MTT working solution is added into each hole, the culture medium is incubated for 3 hours in dark place to induce formazan crystals to form,

Wherein, each 110 mu L of MTT working solution is obtained by mixing 10 mu L of MTT-PBS solution with the concentration of 5 mg/mL with 100 mu L of DMEM culture solution;

(4) After the MTT solution is absorbed and removed, 100 mu L of formazan dissolving solution containing 40% of N, N-dimethylformamide, 16% of sodium dodecyl sulfate and 2% of glacial acetic acid is added into each hole, the solution is subjected to oscillation treatment for 10 minutes to fully dissolve crystals, and an enzyme-labeled instrument is used for measuring absorbance value at the wavelength of 570 nm;

(5) The half inhibition concentration (IC ₅₀) was calculated by a nonlinear regression model with 0.25% DMSO-treated wells absorbance value as negative control (100% cell viability), polymyxin B-treated wells as positive control (0% cell viability), defined as the concentration of compound that reduced cell viability by 50% over the negative control.

As shown in the figure 5, the antibacterial peptide 1-4 has the remarkable advantages that on one hand, the antibacterial peptide has extremely low toxicity to HK-2 human kidney proximal tubule epithelial cells, compared with the clinical polymyxin B, the safety is improved by at least 87 times, compared with the D50 control peptide, the safety is also improved by about 17 times, compared with the non-deuterated antibacterial peptide 5, the safety is improved by about 5.8 times, the risk of damaging normal cells of an organism can be furthest reduced when the antibacterial effect is exerted, the clinical dilemma of high toxicity and low efficiency of the polymyxin B is solved, the antibacterial peptide is expected to become an ideal candidate medicament for replacing the polymyxin B, on the other hand, the low cytotoxicity characteristic is expected to be used for building a better safety basis in anti-infection treatment, effectively supporting the development of novel and safe anti-infection preparations, reducing adverse reactions caused by medicament toxicity in clinical application, improving the safety and tolerance of treatment, and promoting the development of antibacterial medicaments towards safer and more effective directions.

EXAMPLE 5 evaluation of in vivo efficacy of antibacterial peptides

1. Therapeutic efficacy of antibacterial peptides in the rat thigh model of neutropenia mice infected with E.coli ATCC25922

(1) The experiment was performed using CD-1 mice, with 10 (n=10) mice per group. Mice were kept in independent ventilated cages, the indoor temperature was maintained at 24±2 ℃ during the experiment, all mice were free to ingest and drink water, and the mice were randomly assigned to:

0.4 mg/kg polymyxin B treated group, 1.6 mg/kg polymyxin B treated group, 3.2 mg/kg polymyxin B treated group,

0.4 Mg/kg of the D50 control peptide treatment group, 1.6 mg/kg of the D50 control peptide treatment group, 3.2 mg/kg of the D50 control peptide treatment group,

0.4 Mg/kg of the antibacterial peptide 1-treated group, 1.6 mg/kg of the antibacterial peptide 1-treated group, 3.2 mg/kg of the antibacterial peptide 1-treated group,

0.4 Mg/kg of the antibacterial peptide 2-treated group, 1.6 mg/kg of the antibacterial peptide 2-treated group, 3.2 mg/kg of the antibacterial peptide 2-treated group,

0.4 Mg/kg of the antibacterial peptide 3-treated group, 1.6 mg/kg of the antibacterial peptide 3-treated group, 3.2 mg/kg of the antibacterial peptide 3-treated group,

0.4 Mg/kg of the antibacterial peptide 4-treated group, 1.6 mg/kg of the antibacterial peptide 4-treated group, 3.2 mg/kg of the antibacterial peptide 4-treated group,

0.4 Mg/kg of the antibacterial peptide 5-treated group, 1.6 mg/kg of the antibacterial peptide 5-treated group, 3.2 mg/kg of the antibacterial peptide 5-treated group,

A blank group;

(2) On day 4 and day 1 prior to the experiment, cyclophosphamide was injected intraperitoneally in two doses, 150 mg/kg for the first dose and 100 mg/kg for the second dose, to induce a sustained neutrophil status;

(3) All mice were injected with 1×10 ⁵ CFU of standard strain suspension of escherichia coli ATCC25922, respectively, and 1, 3.5, 6 hours after infection, polymyxin B (PMB) or D50 control peptide or antimicrobial peptide was administered by tail vein injection, according to the above group, and saline was administered to the blank group;

(4) Two-sided thigh tissues of all mice were aseptically collected 9 hours after infection, and individual thigh tissue samples were homogenized under ice water bath conditions with PBS at a ratio of 9mL PBS added per gram sample. 1 mL thigh homogenates were quantitatively inoculated onto cystine-dextrose electrolyte deficient (CLED) agar, incubated at 37 ℃ for 24 hours, and bacterial load was determined by Colony Forming Unit (CFU) counting. Log ₁₀ CFU/g was calculated for the reduction in bacterial load between each dosing group and the control group and the results are shown in table 4.

The results are shown in table 4, with antimicrobial peptides 1-4 exhibiting the ability to significantly reduce bacterial load in a mouse escherichia coli ATCC25922 infection model. The bacterial load reduction efficacy of each dose group was improved 9-18 fold compared to polymyxin B, 7-14 fold compared to D50 control peptide, and about 7-10 fold compared to non-deuterated antimicrobial peptide 5. The increasing trend of the logarithmic difference value presentation of the load reduction along with the increase of the dosage shows a clear dosage-effect relationship, and bacteria can be efficiently cleared by low dosage, so that powerful support is provided for developing a novel and efficient preparation for resisting the escherichia coli infection, and the preparation has higher clinical transformation potential.

2. Therapeutic efficacy of antibacterial peptides in the neutropenia mouse thigh model infected with klebsiella pneumoniae ATCC43816

A blank group;

(3) All mice were injected with 1×10 ⁵ CFU klebsiella pneumoniae ATCC43816 standard strain suspension, respectively, into bilateral thigh muscle tissue, and after 2, 6, 10 hours post-infection, polymyxin B (PMB) or D50 control peptide or antimicrobial peptide was administered by tail vein injection, following the above-described group, and saline was administered to the placebo group;

(4) Double-sided thigh tissues of all mice were aseptically collected 16 hours after infection, and individual thigh tissue samples were homogenized under ice water bath conditions with 9mL PBS added per gram of sample. 1mL thigh homogenates were quantitatively inoculated onto cystine-dextrose electrolyte deficient (CLED) agar, incubated at 37 ℃ for 24 hours, and bacterial load was determined by Colony Forming Unit (CFU) counting. Log ₁₀ CFU/g was calculated for the reduction in bacterial load between each dosing group and the control group and the results are shown in table 5.

The results are shown in table 5, with antimicrobial peptides 1-4 exhibiting the ability to significantly reduce bacterial load in a mouse klebsiella pneumoniae ATCC43816 infection model. The bacterial load reduction efficacy of each dose group was improved by 6-11 fold compared to polymyxin B, 3-8 fold compared to D50 control peptide, and about 3.6-7 fold compared to non-deuterated antimicrobial peptide 5. The log difference of the decrease of the bacterial load shows a more stable rising trend along with the gradual increase of the administration dosage, and the relevance of the dosage and the effect is reflected. Of note, a more pronounced antibacterial effect has been demonstrated at lower doses.

3. Therapeutic efficacy of antibacterial peptides in a neutropenic mouse thigh model infected with Acinetobacter baumannii NCTC13424

A blank group;

(3) All mice were inoculated with 1×10 ⁵ CFU acinetobacter baumannii NCTC13424 standard strain suspension by bilateral thigh muscle tissue injection, respectively, and 2, 6, 10 hours post-infection by tail vein injection, polymyxin B (PMB) or D50 control peptide or antimicrobial peptide were administered according to the above group, and saline was administered to the placebo group;

(4) Double-sided thigh tissues of all mice were aseptically collected 16 hours after infection, and individual thigh tissue samples were homogenized under ice water bath conditions with 9mL PBS added per gram of sample. 1mL thigh homogenates were quantitatively inoculated onto cystine-dextrose electrolyte deficient (CLED) agar, incubated at 37 ℃ for 24 hours, and bacterial load was determined by Colony Forming Unit (CFU) counting. Log ₁₀ CFU/g was calculated for the reduction in bacterial load between each dosing group and the control group and the results are shown in table 6.

The results are shown in Table 6, and the antimicrobial peptides 1-4 exhibited excellent ability to reduce bacterial load in the mice Acinetobacter baumannii NCTC13424 infection model. The bacterial load reduction efficacy of each dose group was increased 7.9-14.8 fold compared to polymyxin B, 5.5-7.7 fold compared to D50 control peptide, and about 5-6 fold compared to non-deuterated antimicrobial peptide 5. The log difference of the decrease of the bacterial load shows a steadily rising situation with the gradual increase of the administration dosage, and the association trend of the dosage and the effect is initially shown. Notably, at relatively low doses, a more pronounced antibacterial effect has been observed, and is expected to bring a breakthrough solution for the treatment of acinetobacter baumannii infection.

4. Therapeutic efficacy of antibacterial peptides in the model of the thigh of neutropenic mice infected with Pseudomonas aeruginosa ATCC27853

A blank group;

(3) All mice were inoculated with 1X 10 ⁵ CFU of Pseudomonas aeruginosa ATCC27853 standard strain suspension by bilateral thigh muscle tissue injection, 1, 3.5, 6 hours post infection by tail vein injection, polymyxin B (PMB) or D50 control peptide or antimicrobial peptide, and saline was administered in the blank group according to the above groups;

(4) Two-sided thigh tissues of all mice were aseptically collected 9 hours after infection, and individual thigh tissue samples were homogenized under ice water bath conditions with PBS at a ratio of 9mL PBS added per gram sample. 1 mL thigh homogenates were quantitatively inoculated onto cystine-dextrose electrolyte deficient (CLED) agar, incubated at 37 ℃ for 24 hours, and bacterial load was determined by Colony Forming Unit (CFU) counting. Log ₁₀ CFU/g was calculated for the reduction in bacterial load between each dosing group and the control group and the results are shown in table 7.

The results are shown in Table 7, antimicrobial peptides 1-4 exhibited excellent ability to reduce bacterial load in a model of infection with Pseudomonas aeruginosa ATCC27853 in the rat thigh. The bacterial load reduction efficacy of each dose group was increased 5-11.8 fold compared to polymyxin B, 4-7 fold compared to D50 control peptide, and about 3.9-6.6 fold compared to non-deuterated antimicrobial peptide 5. The logarithmic difference value of the load reduction shows a stable rising trend along with the increase of the dosage, a clear dosage-effect relationship is shown, and the high-efficiency antibacterial effect can be realized with low dosage.

5. Therapeutic efficacy of antibacterial peptides in a neutropenic mouse lung model infected with Acinetobacter baumannii NCTC13424

5 mg/kg of polymyxin B treatment group, 7 mg/kg of polymyxin B treatment group, 14 mg/kg of polymyxin B treatment group,

5 Mg/kg of the D50 control peptide treatment group, 7 mg/kg of the D50 control peptide treatment group, 14 mg/kg of the D50 control peptide treatment group,

5 Mg/kg of the antibacterial peptide 1-treated group, 7 mg/kg of the antibacterial peptide 1-treated group, 14 mg/kg of the antibacterial peptide 1-treated group,

5 Mg/kg of the antibacterial peptide 2-treated group, 7 mg/kg of the antibacterial peptide 2-treated group, 14 mg/kg of the antibacterial peptide 2-treated group,

5 Mg/kg of the antibacterial peptide 3-treated group, 7 mg/kg of the antibacterial peptide 3-treated group, 14 mg/kg of the antibacterial peptide 3-treated group,

5 Mg/kg of the antibacterial peptide 4-treated group, 7 mg/kg of the antibacterial peptide 4-treated group, 14 mg/kg of the antibacterial peptide 4-treated group,

5 Mg/kg of the antibacterial peptide 5-treated group, 7 mg/kg of the antibacterial peptide 5-treated group, 14 mg/kg of the antibacterial peptide 5-treated group,

A blank group;

(2) On day 4 and day 1 prior to the experiment, cyclophosphamide was injected intraperitoneally in two doses, 200 mg/kg for the first dose and 150 mg/kg for the second dose, to induce a sustained neutrophil status;

(3) Mice were inoculated intranasally with a standard bacterial suspension of Acinetobacter baumannii NCTC13424 containing 1X 10 ⁷ CFU/lobe to achieve bilateral lobe precise infection. Polymyxin B (PMB) or D50 control peptide or antimicrobial peptide was administered by subcutaneous injection through the back and neck at 2, 6, 10 hours post-infection, and saline was administered in the placebo group as described above;

(4) Bilateral lung tissues of all mice were aseptically removed 16 hours after infection, and lung tissue samples were homogenized after mixing with PBS at a ratio of 9mL PBS per gram of sample under ice water bath. 1 mL lung tissue homogenates were quantitatively inoculated onto cystine-dextrose-electrolyte-deficient (CLED) agar, incubated at 37 ℃ for 24 hours, and bacterial load was determined by Colony Forming Unit (CFU) counting. Log ₁₀ CFU/g was calculated for the reduction in bacterial load between each dosing group and the control group and the results are shown in table 8.

The results are shown in Table 8, the antimicrobial peptides 1-4 exhibited excellent ability to reduce bacterial load in a mouse lung Acinetobacter baumannii NCTC13424 infection model. The bacterial load reduction efficacy of each dose group was increased 7.9-13.8 fold compared to polymyxin B, 6.5-9 fold compared to D50 control peptide, and about 5.9-7.8 fold compared to non-deuterated antimicrobial peptide 5. The logarithmic difference value of the load reduction shows a stable rising trend along with the increase of the dosage, a clear dosage-effect relationship is shown, and the efficient bacterial removal can be realized by low dosage, so that the method is expected to open up a new path for the treatment of the Acinetobacter baumannii infection in the lung.

6. Therapeutic efficacy of antibacterial peptides in the lung model of neutropenic mice infected with Pseudomonas aeruginosa ATCC27853

A blank group;

(3) Mice were inoculated intranasally with a standard bacterial suspension of Pseudomonas aeruginosa ATCC27853 containing 1X 10 ⁷ CFU/lobe to achieve bilateral lobe precise infection. Polymyxin B (PMB) or D50 control peptide or antimicrobial peptide was administered by subcutaneous injection through the back and neck at 2, 6, 10 hours post-infection, and saline was administered in the placebo group as described above;

(4) Bilateral lung tissues of all mice were aseptically removed 16 hours after infection, and lung tissue samples were homogenized after mixing with PBS at a ratio of 9mL PBS per gram of sample under ice water bath. 1 mL lung tissue homogenates were quantitatively inoculated onto cystine-dextrose-electrolyte-deficient (CLED) agar, incubated at 37 ℃ for 24 hours, and bacterial load was determined by Colony Forming Unit (CFU) counting. Log ₁₀ CFU/g was calculated for the reduction in bacterial load between each dosing group and the control group and the results are shown in table 9.

The results are shown in table 9, with antimicrobial peptides 1-4 exhibiting the ability to significantly reduce bacterial load in a mouse lung pseudomonas aeruginosa ATCC27853 infection model. The bacterial load reduction efficacy of each dose group was 5.9-11.8 fold higher than polymyxin B, 5.5-8.4 fold higher than D50 control peptide, and about 5-6.6 fold higher than non-deuterated antimicrobial peptide 5. The log differences in bacterial load decrease with increasing dose were regularly increasing, clearly showing the dose-effect correlation. The efficient bacterial removal effect can be realized at a low dosage level, and the characteristic provides an important basis for the research and development of novel pseudomonas aeruginosa infection resisting preparations.

7. Therapeutic efficacy of antibacterial peptides in a neutropenic mouse lung model infected with klebsiella pneumoniae ATCC43816

A blank group;

(3) Mice were inoculated intranasally with a standard bacterial suspension of klebsiella pneumoniae ATCC43816 containing 1 x 10 ⁷ CFU/lobe to achieve bilateral lobe precise infection. Polymyxin B (PMB) or D50 control peptide or antimicrobial peptide was administered by subcutaneous injection through the back and neck at 2, 6, 10 hours post-infection, and saline was administered in the placebo group as described above;

(4) Bilateral lung tissues of all mice were aseptically removed 16 hours after infection, and lung tissue samples were homogenized after mixing with PBS at a ratio of 9mL PBS per gram of sample under ice water bath. 1mL lung tissue homogenates were quantitatively inoculated onto cystine-dextrose-electrolyte-deficient (CLED) agar, incubated at 37 ℃ for 24 hours, and bacterial load was determined by Colony Forming Unit (CFU) counting. Log ₁₀ CFU/g was calculated for the reduction in bacterial load between each dosing group and the control group and the results are shown in table 10.

The results are shown in Table 10, the antimicrobial peptides 1-4 exhibited excellent ability to reduce bacterial load in a mouse model of Klebsiella pneumoniae ATCC43816 infection. The bacterial load reduction efficacy of each dose group was increased 7-14 fold compared to polymyxin B, 5-8 fold compared to D50 control peptide, and about 4.8-7 fold compared to non-deuterated antimicrobial peptide 5. The log difference of the bacterial load decrease shows a steady increasing trend with the progressive increase of the dosing dose, which more clearly shows the correlation of the dose and the effect. Notably, at lower doses, a more pronounced bacterial removal effect has been observed, with an outstanding antibacterial effect.

According to the experimental data in tables 8-10, in the lung model of gram-negative bacteria infected neutropenia mice, the antimicrobial peptides 1-4 all showed definite antimicrobial activity in the full dose range, the antibacterial efficacy was linearly enhanced with increasing dose, and typical dose-dependent characteristics were presented. Particularly important, under the same dosage condition, the antibacterial peptide 1-4 has double advantages compared with clinical first-line polymyxin B and control peptide D50, on one hand, the antibacterial effect on the in-vivo antibacterial effect of Acinetobacter baumannii, pseudomonas aeruginosa and klebsiella pneumoniae can be remarkably improved, and the bacterial removal effect can be realized by low dosage, on the other hand, the human kidney proximal tubule epithelial cytotoxicity experiment shows that the IC ₅₀ value of the human kidney proximal tubule epithelial cell is improved by 87 times more than that of the colistin B, the clinical bottleneck of the traditional antibiotic of high toxicity and low efficiency is broken through on the mechanism, and the antibacterial peptide is taken as a novel polypeptide molecule with high-efficiency antibacterial activity and kidney safety, and has definite clinical conversion value in the field of multi-drug resistant gram-negative bacterial infection treatment.

In the specific embodiment of the present invention, the chinese meaning corresponding to the english abbreviation used in the application document is shown in the table:

。

Claims

1. An antibacterial peptide, characterized in that the specific structure is selected from any one of the following (I) - (IV):

(1) As shown in formula (I):

;

(2) As shown in formula (II):

;

(3) As shown in formula (III):

;

(4) As shown in formula (IV):

。

2. A pharmaceutical composition comprising the antibacterial peptide of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient, with or without pharmaceutically acceptable excipients.

3. The pharmaceutical composition of claim 2, wherein the pharmaceutically acceptable excipients comprise one or more of diluents, lubricants, glidants, wetting agents, emulsifiers, pH buffering substances.

4. The pharmaceutical composition according to claim 2, wherein the dosage form of the pharmaceutical composition comprises a tablet, capsule, granule, oral liquid, syrup, powder, pellet, injection, powder injection, suspension, ointment, cream, gel, spray, eye drop, ear drop, nose drop, patch, lotion, suppository, film coating, implant.

5. Use of the antibacterial peptide of claim 1 or the pharmaceutical composition of any one of claims 2-4 for the preparation of a medicament for preventing and/or controlling one or more microbial infections in escherichia coli, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa.