An antimicrobial peptide and its application in pharmacy

Abstract

The present invention relates to a four-arm antimicrobial peptide rich in imidazole groups, wherein the four-arm antimicrobial peptide is composed of four identical polypeptide side chains with 8-12 amino acid residues and three interconnected lysine residues, respectively connected to form amino groups, and terminated with amino groups; each polypeptide side chain is composed of histidine residues, or is composed of histidine residues and one selected from lysine residues or arginine residues. The four-arm antimicrobial peptide of the present invention not only exhibits narrow-spectrum antimicrobial activity to Pseudomonas aeruginosa (P.aeruginosa), but can also be used as a synergistic adjuvant of antibiotics, and more effectively treats skin infections caused by P.aeruginosa without damaging normal human skin microbial flora. At the same time, it has high safety, low hemolytic side effects, and is not easy to develop drug resistance, and has great clinical application potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and in particular relates to an antimicrobial peptide and its application in pharmacy. Background Art

[0002] Skin microbiota plays an important role in human health and disease. When various endogenous and exogenous factors affect the types and numbers of symbiotic microorganisms in the skin, the imbalance of the microbial flora can lead to a variety of skin infectious and autoimmune diseases, such as acne, chronic wound infection, suppurative dermatitis, atopic dermatitis, psoriasis, systemic lupus erythematosus, etc. (Y. Zhu, X. Yu, G. Cheng, Human skin bacterial microbiota homeostasis: A delicate balance between health and disease, mLife 2(2)(2023)107-120). The abuse of topical broad-spectrum antibiotics will change the composition and metabolism of the microbial flora, affect the skin barrier protection and immune function, and thus lead to the occurrence of diseases. Considering the necessity of maintaining the balance of skin bacterial microecology, it is often considered that narrow-spectrum antibiotics are better than broad-spectrum antibiotics in clinical practice, and even broad-spectrum antibiotics should be avoided as much as possible when narrow-spectrum antibiotics can be used (C. Walsh, Where will new antibiotics come from?, Nat Rev Microbiol 1(1)(2003)65-70.). Narrow-spectrum killing ability is therefore gradually considered to be an ideal characteristic of antimicrobial agents, because it can minimize the rapid spread of pathogens and reduce the risk of pathogens becoming resistant to antibiotics, while not harming the microbial flora living in normal human tissues to reduce the risk of secondary infection.

[0003] In recent years, the research on narrow-spectrum antimicrobial agents has mainly focused on small molecule antibiotic compounds, which target specific proteins and pathways of target bacteria, thereby inhibiting or killing specific bacteria. Different from the bactericidal mechanism of traditional antibiotics, antimicrobial peptides mainly interact with bacterial cell membranes and cause membrane perturbations, thereby producing direct membrane-breaking and bactericidal effects (Y. Shi, XQ Feng, LMLin, J. Wang, JY Chi, BY Wu, GL Zhou, FY Yu, Q. Xu, DJ Liu, GL Quan, C. Lu, X. Pan, JF Cai, CB Wu, Virus-inspired surface-nanoengineered antimicrobial liposome: A potential system to simultaneously achieve high activity and selectivity, Bioact Mater 6 (10) (2021) 3207-3217). Because antimicrobial peptides can bypass the resistance mechanism of bacteria to existing antibiotics, have high bactericidal efficiency and are not easy to develop resistance, they are considered to be one of the most promising alternatives to antibiotics. However, so far, there are still few examples of successful development and clinical application of antimicrobial peptides (or polymers) that can achieve narrow-spectrum antibacterial activity (YMWu, K.Chen, J.ZWang, MZChen, Y.Chen, YRShe, Z.Yan, RHLiu, Host defense peptide mimicking antimicrobial amino acid polymers and beyond: Design, synthesis and biomedical applications, Prog Polym Sci 141 (2023)).

[0004] The structure of natural antimicrobial peptides is usually composed of 12-50 amino acids, including cationic hydrophilic regions and hydrophobic regions. The amino acid composition of these peptides includes more than 2 basic amino acids (such as lysine and arginine), and about 50% hydrophobic amino acids (such as tryptophan, leucine and alanine). Regarding the model of the action of antimicrobial peptides on the membrane surface, various models have been proposed, including the barrel-board model, the carpet model and the annular pore model. These models emphasize that the way antimicrobial peptides kill bacteria generally depends on the electrostatic interaction between their own positively charged residues and the negatively charged bacterial cell membrane. After the antimicrobial peptides electrostatically bind to the bacterial cell membrane, they can aggregate on the membrane surface, further induce the disturbance or lysis of the bacterial cell membrane, and finally kill the bacteria. It is worth noting that the composition of bacterial cell membranes and mammalian cell membranes is significantly different. The outermost leaflet of the bacterial cell membrane contains a high proportion of phospholipids with negatively charged head groups, and the bacterial cell wall also contains a large amount of negatively charged teichoic acid and lipopolysaccharide, while the mammalian cell membrane is mainly composed of neutral lipids. Therefore, antimicrobial peptides can selectively bind to negatively charged bacterial pathogens, while having little effect on mammalian cells (LMLin, JYChi, YLYan, R.Luo, XQFeng, YWZheng, DYXian, X.Li, GLQuan, DJLiu, CBWu, C.Lu, X.Pan, Membrane-disruptive peptides / peptidomimetics-basedtherapeutics: Promising systems to combat bacteria and cancer in the drug-resistant era, Acta Pharm Sin B11(9)(2021)2609-2644.).

[0005] Although natural antimicrobial peptides have excellent performance in membrane-breaking and bactericidal effects, their application is limited by high toxicity, low proteolytic stability and high manufacturing costs. Therefore, in order to mimic the structural and functional properties of natural antimicrobial peptides, including amphipathic structure, positive charge, secondary structure and amino acid sequence, many new artificial antimicrobial peptides or antimicrobial polymers have been carefully designed and synthesized. Compared with natural antimicrobial peptides, they usually have more stable physicochemical properties, stronger antimicrobial activity and higher selectivity.

[0006] The positive charge and hydrophobicity of antimicrobial peptides have a key influence on their antimicrobial activity. Studies have shown that increasing the proportion of hydrophobic parts in the structure can enhance the penetration of antimicrobial peptides into bacterial cell membranes, but it also increases the possibility of binding to mammalian cell membranes with neutral surface charge, resulting in increased hemolytic side effects and reduced selectivity. In recent years, studies have successfully constructed a purely hydrophilic star-shaped polylysine antimicrobial peptide through molecular structure design. This unique design controls the proportion of the hydrophobic part of the antimicrobial peptide within a certain range, while increasing the number and density of positive charges, breaking the limitation of the low charge density of traditional linear antimicrobial peptides, and ultimately achieving improved membrane penetration efficiency, antibacterial activity and selectivity, while reducing hemolytic side effects (C. Lu, GL Quan, M. Su, A. Nimmagadda, WD Chen, M. Pan, P. Teng, FY Yu, X. Liu, L. Jiang, WY Du, W. Hu, F. Yao, X. Pan, CB Wu, DJ Liu, JF Cai, Molecular Architecture and Charging Effects Enhance the In Vitro and In Vivo Performance of Multi-Arm Antimicrobial Agents Based on Star-Shaped Poly (L-lysine), Adv Ther 2 (12) (2019).). This type of antimicrobial peptide with a multi-arm structure shows great application potential in binding to negatively charged bacterial cell membranes, and its safety is also relatively high. However, this design and activity can only be reflected in the antimicrobial peptide killing bacteria but not animal cells (selectivity). The specific killing effect on specific strains (narrow-spectrum antimicrobial activity) is not obvious.

[0007] The outermost leaflet of the bilayer contains a high proportion of phospholipids with negatively charged head groups, which is a common feature of bacterial cytoplasmic membranes. However, the composition of the phospholipid tails of different bacterial cell membranes is actually very different, which suggests that by exploiting these differences to design antimicrobial peptides, it is possible to obtain new narrow-spectrum antimicrobial agents that are effective against specific strains. It is reported that the cell membranes of Gram-negative bacteria such as Pseudomonas aeruginosa are rich in unsaturated phosphatidylethanolamine, while the cell membranes of Gram-positive bacteria such as Staphylococcus are mainly composed of phosphatidylglycerol and cardiolipin. The content of unsaturated phospholipids in the cell membrane of P. aeruginosa is often higher than that of saturated phospholipids, while the cell membranes of other commensal bacteria in human tissues (such as Staphylococcus) have a higher proportion of saturated phospholipids.

[0008] In recent years, some studies have pointed out that antimicrobial peptides have the potential to serve as synergistic adjuvants for antibiotics, which can synergize with antibiotics to enhance the antibacterial effect. The combined use of antimicrobial peptides and antibiotics can help improve the efficacy of antibiotics while reducing the dosage used, thereby reducing possible toxic side effects. Generally, antimicrobial peptides work by destroying bacterial cell membranes. They can increase the permeability of bacterial cell membranes, help antibiotics better target the inside of bacteria, and ultimately exert a synergistic antibacterial effect. Wong et al. (Z.Shao, E.Wulandari, RCLin, J.Xu, K.Liang, EHWong, Two plus one: combination therapy tri-systems involving two membrane-disruptingantimicrobial macromolecules and antibiotics, ACS Infectious Diseases 8(8)(2022)1480-1490) described a new "three-system" combination therapy system for multidrug-resistant bacteria, including synthetic cationic antimicrobial polymers, colistin mesylate, and doxycycline. The results showed that compared with single antibiotic treatment, the system exhibited stronger bactericidal activity, and the number of Pseudomonas aeruginosa could be reduced to below 99.999% in just 3 hours at a low dose. In the study of Yang's team, they found that a guanidine-functionalized polycarbonate pEt_20 with a degree of polymerization of 20 was used in combination with rifampicin to show excellent synergistic antibacterial effects (X. Ding, C. Yang, W. Moreira, P. Yuan, B. Periaswamy, PF de Sessions, H. Zhao, J. Tan, A. Lee, KX Ong, A macromolecule reversing antibiotic resistance phenotype and repurposing drugs as potent antibiotics, Advanced Science 7 (17) (2020) 2001374.). In the MDR baumannii bacteremia model of mice, pEt_20 combined with antibiotics showed more significant bacterial clearance and higher mouse survival rate.

[0009] Given the complexity of biotechnology and drug efficacy, researchers are working hard to screen suitable antimicrobial peptides for clinical applications that can effectively inhibit bacteria and be used in combination with antibiotics. Summary of the invention

[0010] Based on this, the purpose of the present invention is to provide a series of multi-arm antimicrobial peptides containing imidazole groups, which have narrow-spectrum antimicrobial activity and can effectively inhibit the growth of bacteria as antibiotic synergists, especially the growth of Pseudomonas aeruginosa (P. aeruginosa). The antimicrobial peptides and antibiotics co-loaded drugs have significant antibacterial effects, are less likely to develop drug resistance, and have clinical application value.

[0011] The technical solutions to achieve the above purpose include the following:

[0012] The first aspect of the present invention is to provide a four-arm antimicrobial peptide, which is composed of four identical polypeptide side chains with 8-12 amino acid residues connected to the amino groups of three interconnected lysine residues, and terminated with amino groups; each polypeptide side chain is composed of a histidine residue, or a histidine residue and one selected from a lysine residue or an arginine residue.

[0013] Or the four-arm antimicrobial peptide has the following structural formula,

[0014]

[0015] When R1 represents the side chain of a histidine residue, n = 8-12;

[0016] Or when R1 represents a combination of a lysine residue side chain and a histidine residue side chain, or a combination of an arginine residue side chain and a histidine residue side chain, n=4-6.

[0017] In some embodiments, the polypeptide side chain composition is: a combination of lysine residues and histidine residues, or a combination of arginine residues and histidine residues.

[0018] In some embodiments, the polypeptide side chain consists of 8-12 histidine residues, more preferably 9-11 histidine residues, such as 9, 10, or 11 histidine residues.

[0019] In some embodiments, the polypeptide side chain is at least one of (KHKHKHKHKH)4K2K-NH2, (RHRHRHRHRH)4K2K-NH2 or (HHHHHHHHHH)4K2K-NH2.

[0020] In some embodiments, the four-arm antimicrobial peptide is (HHHHHHHHHH)4K2K-NH2.

[0021] The second aspect of the present invention is to provide the use of the above four-arm antimicrobial peptide in the preparation of drugs against Gram-negative bacteria.

[0022] The third aspect of the present invention is to provide the use of the above four-arm antimicrobial peptide as a synergistic adjuvant of antibiotics in combination with antibiotics in the preparation of antibacterial drugs.

[0023] The fourth aspect of the present invention is to provide an antibacterial drug comprising the above-mentioned four-arm antibacterial peptide and pharmaceutically acceptable excipients.

[0024] The fifth aspect of the present invention is to provide an antibacterial combination drug, the active ingredients of which include the above-mentioned four-arm antimicrobial peptide and antibacterial antibiotics, the four-arm antimicrobial peptide and antibiotics respectively becoming independent dosing units, or the four-arm antimicrobial peptide and antibiotics together forming a combined dosing unit.

[0025] In some embodiments, the bacteria are Gram-negative bacteria, and further, the Gram-negative bacteria are Acinetobacter baumannii or Pseudomonas aeruginosa.

[0026] In some preferred embodiments, the antibiotic is a tetracycline antibiotic, preferably doxycycline.

[0027] The mass ratio of the four-arm antimicrobial peptide to doxycycline is (6-10):1, more preferably (7-9):1, and more preferably the dosage form is a thermosensitive hydrogel (TSG).

[0028] In some embodiments, the dosage form of the drug is selected from at least one of ointment, cream, paste, gel, solution lotion, film, injection, aerosol or transdermal patch.

[0029] In some embodiments, the drug is in the form of at least one of an ointment, a hydrogel or an injection.

[0030] The present invention designs and screens a series of four-arm antimicrobial peptides rich in imidazole groups. These antimicrobial peptides are composed of lysine / histidine, arginine / histidine or histidine, and their structures are designed to be four-arm structures, which are conducive to increasing the density of imidazole groups in the antimicrobial peptide structure and enhancing its selectivity and narrow-spectrum antimicrobial activity. In particular, when the four-arm antimicrobial peptide is 4H10, the four-arm antimicrobial peptide can be used as an antibiotic synergist, and without damaging the normal skin microbial flora of the human body, it can more effectively play a synergistic antibacterial role in treating skin infections caused by Pseudomonas aeruginosa (P. aeruginosa), while reducing the hemolytic side effect and being less likely to produce antibiotic resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is 4H10 in D2O 1 H NMR spectra.

[0032] Figure 2MIC values ​​of various antimicrobial peptides against different bacteria (a) and different clinical Pseudomonas aeruginosa strains (b).

[0033] Figure 3 Hemolytic effect of various antimicrobial peptides.

[0034] Figure 4 It is the selectivity index of various antimicrobial peptides against multiple clinical Pseudomonas aeruginosa strains.

[0035] Figure 5 This is a checkerboard dilution experiment of 4H10 and doxycycline against Pseudomonas aeruginosa; the darker the module color in the figure, the smaller the inhibitory effect on bacterial growth; the black and yellow balls represent the MIC values ​​of 4H10 and doxycycline, respectively; the white balls indicate the concentrations at which the two have a synergistic effect.

[0036] Figure 6 TEM image of Pseudomonas aeruginosa treated with 4H10 and doxycycline.

[0037] Figure 7 The resistance development of P. aeruginosa to 4H10 (0.5×MIC) and doxycycline (0.5×MIC) after 50 passages.

[0038] Figure 8 Skin abscesses (a) and live bacterial counts in tissues (b) after treatment of Pseudomonas aeruginosa infections with PBS and 4H10 / Doxycyline TSG.

[0039] Fig. 9 Schematic diagram of the preparation process of the imidazole-rich four-arm antimicrobial peptide, in which the gray balls represent the solid phase resin used for solid phase synthesis. DETAILED DESCRIPTION

[0040] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0041] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0042] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0043] Definitions: To facilitate understanding of this technology, certain terms and phrases are defined below.

[0044] MIC: The mixture of MHB and bacterial solution was used as the positive control, and pure MHB culture medium was used as the negative control. The minimum concentration that inhibited bacterial growth was defined as the minimum inhibitory concentration.

[0045] The imidazole group is a five-membered heterocyclic group containing two nitrogen atoms and three carbon atoms. In the imidazole ring, the two nitrogen atoms are located at the 1 and 3 positions of the ring, and they are adjacent and alternate with the carbon atoms. The molecular formula of the imidazole group is C3H3N2.

[0046] K:Lysine

[0047] H:Histidine

[0048] R:Arginine.

[0049] Doxycycline: also known as doxycycline hydrochloride. English name: Doxycycline Hyclate.

[0050] The present invention is further described in detail below with reference to specific embodiments.

[0051] Example 1

[0052] In this embodiment, a four-arm antimicrobial peptide based on histidine is experimentally confirmed to be beneficial for increasing the density of imidazole groups in the antimicrobial peptide structure, thereby enhancing its selectivity and achieving narrow-spectrum antibacterial properties.

[0053] ①Synthesis and characterization of four-arm antimicrobial peptides rich in imidazole groups

[0054] A series of imidazole-rich antimicrobial peptides were designed, including (KHKHKHKHKH)4K2K-NH2(4K5H5), (RHRHRHRHRH)4K2K-NH2(4R5H5), and (HHHHHHHHHH)4K2K-NH2(4H10).

[0055] A series of imidazole-rich four-arm antimicrobial peptides based on lysine / histidine, arginine / histidine and histidine were synthesized on Fmoc-Rink Amide MBHA resin using the standard Fmoc solid-phase peptide synthesis strategy.

[0056] The specific synthesis steps are as follows: Fig. 9 Schematic diagram of the preparation process of a series of imidazole-rich four-arm antimicrobial peptides, the gray ball represents the solid phase resin used for solid phase synthesis):

[0057] S1. Remove the Fmoc protecting group on the resin with 20% piperidine DMF solution (×2), and then wash the resin three times with DCM and DMF. Use four times the equivalent of Fmoc-Lys(Fmoc)-OH to couple with hydroxybenzotriazole (HOBt) and N,N′-diisopropylcarbodiimide (DIC) in a solid phase synthesis tube for 4 hours.

[0058] S2. The resin was washed three times with DCM and DMF, and Fmoc deprotection was performed with 20% piperidine DMF solution (×2). The second Fmoc-Lys(Fmoc)-OH was coupled to the resin according to the above scheme to obtain the second generation of lysine dendrimers.

[0059] S3. The resin is repeatedly subjected to Fmoc deprotection and subsequent amino acid coupling, and reacted with 12 equivalents of Fmoc-His(Trt)-OH, Fmoc-Lys(Fmoc)-OH or Fmoc-Arg(Pbf)-OH until different target peptides are obtained.

[0060] S4. After the peptide was deprotected, it was cleaved from the resin using a mixed solution of 92.5% (v / v) trifluoroacetic acid (TFA), 2.5% triethylsilane (TIS), 2.5% ethanedithiol (EDT) and 2.5% H2O, with shaking reaction at room temperature for 2.5 h.

[0061] S5. All crude products were precipitated in cold ether, purified, and finally analyzed using a Shimadzu LC-20A HPLC system, MALDI-TOF and 1 H NMR characterization.

[0062] The results are shown in Table 1 and Figure 1 The target product was analyzed by high performance liquid chromatography (HPLC), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and 1 H NMR confirmed their purity and structure, indicating that the target products were successfully synthesized and the designed synthetic method was feasible.

[0063] Table 1 HPLC and ESI-MS characterization of antimicrobial peptides

[0064]

[0065] ②In vitro and in vivo antibacterial activity and selectivity studies

[0066] a) Minimum inhibitory concentration study

[0067] In order to further study the antibacterial effects of 4K5H5, 4R5H5 and 4H10, the minimum inhibitory concentration (MIC) was determined for a variety of pathogenic microorganisms that are of great significance in clinical medicine, including methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli (E. coli), Acinetobacter baumannii (A. baumannii) and Pseudomonas aeruginosa (P. aeruginosa).

[0068] Gram-positive bacteria (methicillin-resistant Staphylococcus aureus (MRSA, ATCC 33591)), Gram-negative bacteria (Escherichia coli (E. coli, ATCC 25922), Acinetobacter baumannii (A. baumannii, ATCC 19606), Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853)) were inoculated in MHB and cultured to the logarithmic growth phase, and then diluted to 2×105 CFU·mL -1 The antimicrobial peptide samples were dissolved in phosphate buffered saline (PBS) and diluted to 200, 100, 50, 25, 12.5, 6.2, 3.1, 1.55, and 0.78 μg mL -1 . Add 50 μL of antimicrobial peptide solution of different concentrations to the sample well, and then add 50 μL of diluted bacterial solution. The mixed sample is placed in a 37°C incubator for 16 hours. Measure the absorbance at 600 nm with an enzyme-labeled instrument and record the experimental results.

[0069] The three imidazole-rich four-arm antimicrobial peptides synthesized in the above method ① were tested for their effects on different bacteria ( Figure 2 From the MIC values ​​in a), it can be seen that they exhibit a narrow spectrum antibacterial activity against the standard strain of Pseudomonas aeruginosa. Their MIC values ​​against the standard strain of Pseudomonas aeruginosa are more than 4 times lower than the MICs of other pathogens.

[0070] We further evaluated the antibacterial effects of the imidazole-rich four-arm antimicrobial peptides (4K5H5, 4R5H5 and 4H10) against various clinically isolated Pseudomonas aeruginosa strains. Figure 2 As shown in b, different clinical Pseudomonas aeruginosa strains ( Figure 2b) showed a certain degree of difference in sensitivity to 4K5H5, 4R5H5 and 4H10. Among them, 4H10 had the lowest MIC value for all tested Pseudomonas aeruginosa strains, with a geometric mean (Gm) of 4μM. The results showed that by designing the antimicrobial peptide into a multi-arm structure, the density of the imidazole group can be effectively increased, thereby obtaining a narrow-spectrum antimicrobial peptide with a targeted bactericidal effect on Pseudomonas aeruginosa. In addition, our study also found that when the imidazole group in the 4H10 antimicrobial peptide structure was replaced with an amino or guanidine group (i.e., 4K5H5 and 4R5H5), the activity of the antimicrobial peptide against various clinically isolated Pseudomonas aeruginosa strains would decrease, which further highlights the key role of the imidazole group in achieving specific bactericidal effects on Pseudomonas aeruginosa, which is beneficial to improve the specificity of the drug.

[0071] Example 2: Hemolytic activity study

[0072] Since high hemolysis rate is one of the main reasons limiting the application of many antimicrobial peptides, we used rat erythrocytes to perform hemolysis experiments to evaluate the hemolytic activity of these antimicrobial peptides.

[0073] Fresh rat red blood cells (rRBC) treated with EDTA-K2 were collected by centrifugation. The red blood cells were washed three times with PBS and prepared into a 5% (v / v) red blood cell suspension. The concentrations of 2000, 1000, 500, 250, 125, 62.5 and 31.3 μg·mL were prepared with PBS. -1 The antimicrobial peptide solution was then added with an equal volume of red blood cell suspension and incubated in a 37°C incubator for 1 h.

[0074] In this experiment, PBS and 2% Triton X-100 treated RBC suspension were used as negative and positive controls, respectively. After the incubation, centrifuge at 1000×g for 10 min. Dilute 30 μL of supernatant with 100 μL PBS in another 96-well plate, and then measure the absorbance value at 540 nm using a microplate reader. The hemolysis rate of each sample was calculated according to the following formula:

[0075] Hemolysis rate (%) = (A sample -A PBS ) / (A TritonX-100 -A PBS )×100%

[0076] A sample It represents the absorbance of the mixture of antimicrobial peptide and red blood cells;

[0077] A TritonX-100 represents the absorbance of the positive control;

[0078] A PBS Indicates the absorbance of the negative control.

[0079] The results are as follows Figure 3 As shown in Figure 2, 4K5H5, 4R5H5, and 4H10 all showed negligible hemolytic activity, and their HC5 (i.e., the drug concentration required to cause 5% hemolysis of erythrocytes) was greater than 1000 μg mL -1 The results show that the series of imidazole-rich four-arm antimicrobial peptides (4K5H5, 4R5H5 and 4H10) designed and synthesized by us have good biosafety and application potential.

[0080] Subsequently, we calculated the ratio of HC5 to Gm of different antimicrobial peptides to obtain the selectivity index to evaluate the selectivity of this series of imidazole-rich four-arm antimicrobial peptides against multiple clinical Pseudomonas aeruginosa strains.

[0081] like Figure 4 As shown in the figure, 4H10 has the highest selectivity index of 40.0, while the selectivity indexes of 4R5H5 and 4K5H5 are 17.2 and 12.6, respectively. According to literature reports, the selectivity index of antibacterial drugs needs to be higher than 10 to have good clinical application and research value. Among this series of antimicrobial peptides, 4H10, which contains the highest density of imidazole groups in its structure, exhibits the strongest antibacterial activity and the highest selectivity against Pseudomonas aeruginosa. Therefore, this result once again proves that the imidazole group has a unique role in achieving specific killing of Pseudomonas aeruginosa.

[0082] Example 3 In vitro antibacterial activity and mechanism study of antimicrobial peptides combined with antibiotics

[0083] a) In vitro combined antibacterial activity study

[0084] It is reported that the combination of antimicrobial peptides and antibiotics is beneficial to enhance the efficacy of antibiotics and reduce the dosage, thereby reducing the toxic side effects that may be caused by the large-scale use of antibiotics. Therefore, in order to better deal with Pseudomonas aeruginosa infections in practical applications, we further studied the combined antibacterial ability of 4H10 and a variety of broad-spectrum or traditional antibiotics targeting Gram-negative bacteria. The checkerboard method was used to evaluate the combined antibacterial effect of 4H10 and a variety of commercial antibiotics, and the partial inhibitory concentration index (FICI) was calculated.

[0085] The chessboard method was used to study the interaction between antimicrobial peptides and different antibiotic combinations. Pseudomonas aeruginosa was cultured to the logarithmic growth phase and diluted to 2 × 10 in MHB. 5 CFU·mL -1. Prepare gradient dilutions (2-fold) of 4H10 and antibiotics ranging from 0 to 4×MIC. Add 25 μL of 4H10 solution to the columns of a 96-well plate and an equal volume of antibiotic solution to the rows. Then, add 50 μL of the diluted bacterial solution to each sample well. The mixed samples were placed in a 37°C incubator and cultured for 16 h. Measure the absorbance at 600 nm with a microplate reader, and record the MIC when 4H10 and antibiotics were administered alone or in combination. The partial inhibitory concentration index (FICI) was calculated according to the following formula:

[0086]

[0087] Among them, MICA,alone and MICB,alone are the MICs of 4H10 and antibiotics when acting alone, MICA,combination and MICB,combination are the MICs of antibiotics and 4H10 when used in combination. FICI ≤ 0.5 indicates synergistic effect (S), > 0.5 but < 1 indicates partial synergistic effect (PS), and > 1 but < 4 indicates additive effect (A).

[0088] The experimental results are as follows Figure 5 As shown, 4H10 and doxycycline have obvious synergistic antibacterial effects (FICI≤0.5), the FICI value is calculated to be 0.375, and the mass ratio of 8:1 (4H10: doxycycline) is the optimal ratio for the combination of the two. This result preliminarily indicates that this imidazole-rich four-arm antimicrobial peptide has good application potential for development as an antibiotic synergistic adjuvant.

[0089] like Figure 5 Shown are the results of a checkerboard dilution experiment of 4H10 and doxycycline against Pseudomonas aeruginosa; Figure 5 The darker the color of the middle module, the smaller the inhibitory effect on bacterial growth; the black and yellow balls represent the MIC values ​​of 4H10 and doxycycline, respectively; the white balls indicate the concentrations at which the two have a synergistic effect.

[0090] b) Study on antibacterial mechanism

[0091] To elucidate the mechanism of antibiotic enhancement by 4H10, we used transmission electron microscopy (TEM) imaging to evaluate the changes in the cell morphology of P. aeruginosa after treatment with 4H10 combined with doxycycline.

[0092] TEM was used to observe the morphological changes of Pseudomonas aeruginosa before and after treatment with different drugs. The bacteria were cultured to the logarithmic growth phase, collected and washed three times with PBS. Then, 800 μL of bacterial suspension was added to an equal volume of a mixed solution containing 4H10 and doxycycline (100 μg mL -1 4H10 and 25 μg mL -1Doxycycline). Bacteria treated with PBS served as the control group. After incubation at 37°C for 4 hours, the bacterial pellet was centrifuged at 5000×g for 15 minutes, washed with PBS, and collected. 1 mL of 2.5% glutaraldehyde solution was added to the bacterial pellet and blown evenly, and fixed overnight at 4°C. The bacterial pellet was washed with PBS and then further fixed with 1% osmium acid for 1 hour. After washing with PBS, the samples were then dehydrated with different concentrations of ethanol (30, 50, 70, 80, 90, and 95%), and then the dehydrated samples were transferred to acetone for 20 minutes, mixed with embedding medium and acetone (V / V=1 / 1), treated for 1 hour, and then treated with a mixture of embedding medium and acetone (V / V=3 / 1) for 3 hours, and finally incubated in Spurr resin overnight. The sample sections were stained and the bacterial morphology was observed by TEM.

[0093] like Figure 6 As shown in the figure, we observed that the outer and inner membranes of the bacterial cells treated with 4H10 and doxycycline were significantly damaged, which provided conditions for the increase of doxycycline concentration in the cells. The results showed that this imidazole-rich four-arm antimicrobial peptide may increase the permeability of the Pseudomonas aeruginosa cell membrane, promote the enrichment of doxycycline to the intracellular targets of the bacteria, and ultimately exert a synergistic antibacterial effect.

[0094] ④ In vitro drug resistance induction study

[0095] In order to study whether 4H10 is likely to cause bacterial resistance, we used the in vitro drug concentration increase method to induce resistance in Pseudomonas aeruginosa and determined the changes in MIC values ​​by the broth microdilution method.

[0096] Pseudomonas aeruginosa was inoculated in MHB and cultured to the logarithmic growth phase. 50 μL of bacterial solution was added to MHB containing antibiotics (1 / 2×MIC) and cultured in a 37°C incubator for 10 hours. Every 10 hours is one generation, and the strain is continuously transferred and cultured until the strain can grow stably in MHB broth with 1 / 2MIC antibiotics. If the MIC value after induction is higher than the original MIC value, it means that the bacterial solution is resistant to antibiotics or antimicrobial peptoids, and then a new MHB containing antibiotics (1 / 2×MIC) is used to continue induction. After each antibiotic concentration induction is completed, continue to culture in MHB for three generations to ensure the stability of drug resistance induction. Then measure the MIC values ​​of antimicrobial peptides and doxycycline. If the MIC value is higher than the initial measurement value, it indicates that the bacteria have begun to develop drug resistance.

[0097] The development of resistance of Pseudomonas aeruginosa to 4H10 (0.5×MIC) and doxycycline (0.5×MIC) after 50 passages Figure 7As shown in Figure 2, after 50 generations of induction, Pseudomonas aeruginosa had developed resistance to doxycycline, and its MIC value increased by 64 times (reaching 400 μg mL -1 ). However, in contrast, the MIC value of 4H10 against Pseudomonas aeruginosa did not change. This result further suggests that this imidazole-rich four-arm antimicrobial peptide may have a unique advantage in killing drug-resistant bacteria.

[0098] ⑤Study on the treatment of mouse skin infection

[0099] In order to improve the local retention of 4H10 and doxycycline in the body and facilitate injection administration, we further constructed a thermosensitive hydrogel (TSG) for subsequent in vivo efficacy evaluation experiments.

[0100] a) Construction and treatment of skin infection model

[0101] Healthy ICR mice weighing 18 to 22 g were used. Pseudomonas aeruginosa in the logarithmic growth phase was washed with sterile PBS and resuspended to 1×10 8 CFU·mL -1 , and then 50 μL was injected subcutaneously into the mice. After the bacterial solution was basically absorbed (30 min), 100 μL of a certain concentration of 4H10 / Doxycyline TSG (mass ratio of 8:1) precursor solution or pure PBS solution (as a positive control) was injected into the same modeling site. After 48 h, the mice were killed by cervical dislocation, and the skin of the subcutaneous abscess on the back was dissected.

[0102] b) Subcutaneous live bacteria content analysis

[0103] Add 1 mL of sterile PBS to make a homogenate of the skin sample. Dilute the homogenate in a 10-fold gradient to make dilutions of different concentrations. Take 10 μL of the dilution and inoculate it on an MH agar plate, incubate it at 37°C overnight, and count the number of bacterial colonies on the plate.

[0104] The number of viable bacteria in each milliliter of homogenate = number of colonies × 100 × dilution factor.

[0105] One-way analysis of variance (ANOVA) was used for statistical analysis, and the LSD-t method was used for pairwise comparisons between groups. P<0.05 was considered to be significantly different, and the levels of significant difference were expressed as follows: *p<0.05, **p<0.01, ***p<0.001.

[0106] In this part of the experiment, we established a mouse deep skin infection model caused by Pseudomonas aeruginosa and selected a commercially available antibiotic ointment as a control. Figure 8 As shown, mice were injected subcutaneously with 1×108 CFU·mL -1After 48 hours of inoculation with Pseudomonas aeruginosa, the backs of mice in the control group were obviously swollen, and large abscesses were observed under the skin, indicating that the model was successfully established. After treatment with thermosensitive hydrogel co-loaded with 4H10 and doxycyline (4H10 / Doxycyline TSG), the subcutaneous abscesses were significantly reduced, indicating that the combined treatment has a certain antibacterial effect.

[0107] To further determine the number of viable bacteria in the subcutaneous tissue, we took tissue samples from the skin abscess area and added them to sterile PBS to make a homogenate. After 10-fold dilution, they were cultured on MH agar plates and quantitatively analyzed.

[0108] PBS and 4H10 / Doxycyline TSG in the treatment of skin abscesses caused by Pseudomonas aeruginosa and the content of live bacteria in tissues Figure 8 As shown, it can be seen that the use of 4H10 / Doxycyline TSG can effectively eliminate up to 97.1% of Pseudomonas aeruginosa in mouse skin within two days. Compared with the commercially available ointment group, its clearance rate is more than 12 times higher (p<0.001), indicating that it has a significant antibacterial effect in vivo. These research results preliminarily demonstrate the advantages of 4H10 combined with doxycycline in in vivo antibacterial effects, suggesting that this imidazole-rich four-arm antimicrobial peptide has great potential for clinical application.

[0109] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A four-arm antimicrobial peptide, characterized in that: The four-arm antimicrobial peptide is composed of four identical polypeptide side chains with 8-12 amino acid residues connected to the amino groups of three interconnected lysine residues, and is terminated with amino groups; the four-arm antimicrobial peptide is at least one of (KHKHKHKHKH)4K2K-NH2, (RHRHRHRHRH)4K2K-NH2 or (HHHHHHHHHH)4K2K-NH2; the four-arm antimicrobial peptide has the following structural formula, R1 represents the side chain of a histidine residue; or R1 represents a combination of the side chains of a lysine residue and a histidine residue, or a combination of the side chains of an arginine residue and a histidine residue.

2. The four-arm antimicrobial peptide according to claim 1, characterized in that The four-arm antimicrobial peptide is (HHHHHHHHHH)4K2K-NH2.

3. Use of the four-arm antimicrobial peptide according to any one of claims 1 to 2 in the preparation of drugs against Gram-negative bacteria, wherein the Gram-negative bacteria is Pseudomonas aeruginosa.

4. Use of the four-arm antimicrobial peptide according to any one of claims 1-2 as an antibiotic synergistic adjuvant in combination with antibiotics in the preparation of antibacterial drugs, wherein the bacteria is Pseudomonas aeruginosa and the antibiotic is doxycycline.

5. An antibacterial drug, characterized in that: The active ingredient of the antibacterial drug comprises the four-arm antibacterial peptide according to any one of claims 1 to 2, and pharmaceutically acceptable excipients.

6. An antibacterial combined drug, characterized in that: Its active ingredients include the four-arm antimicrobial peptide according to any one of claims 1 to 2 and an antibacterial antibiotic, wherein the four-arm antimicrobial peptide and the antibiotic respectively form independent dosing units, or the four-arm antimicrobial peptide and the antibiotic together form a combined dosing unit, and the antibiotic is doxycycline.

7. The antibacterial combination drug according to claim 6, characterized in that: The mass ratio of the four-arm antimicrobial peptide to doxycycline is (6-10):

1.

8. The antibacterial combination drug according to any one of claims 6 to 7, characterized in that: The dosage form of the drug is a thermosensitive hydrogel.

9. The antibacterial combination drug according to any one of claims 6 to 7, characterized in that: The dosage form of the drug is selected from one of ointment, cream, paste, gel, solution lotion, film coating, injection, aerosol or transdermal patch.

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