Lugdunin-based novel cationic cyclopeptide antibiotics

Abstract

The invention discloses a cationic cyclopeptide antibiotic based on Lugdunin and a preparation method of the cationic cyclopeptide antibiotic. The cationic cyclopeptide antibiotic provided by the invention has a membrane destroying effect of a cationic antibacterial peptide, is easy to synthesize, and has better clinical application potential in resisting drug-resistant bacterial infection.

Description

Technical Field

[0001] The invention belongs to the field of organic chemistry, and in particular relates to a novel cationic cyclic peptide antibiotic based on Lugdunin. Background Art

[0002] With the increasing prevalence of drug-resistant bacterial infections, antibiotic resistance has gradually become a major challenge to human health and medical practice. Drug-resistant bacteria will make some common infectious diseases that have been easily cured with traditional antibiotics since the discovery of penicillin difficult to treat again. In addition to increasing the risk of treatment failure and complications, drug-resistant bacterial infections will also lead to an increase in mortality from infection-related diseases. Currently, about 700,000 patients die each year from drug-resistant bacterial infections. If no effective antibacterial drugs are discovered, this number will increase to 10 million per year by 2050, with direct economic losses exceeding US$10 billion. In addition, the widespread spread of drug-resistant bacteria will make modern medical procedures such as surgery and organ transplantation vulnerable to infection, thereby affecting the implementation of these procedures and even hindering the development of medical technology. However, due to the low commercial returns of antimicrobial agents at present, companies lack enthusiasm and investment in researching antimicrobial agents, resulting in a serious shortage of antimicrobial pipelines. Therefore, it is more urgent than ever to develop new antimicrobial agents or strategies to combat drug-resistant bacterial infections.

[0003] Lugdunin is the first cyclopeptide antibiotic containing a thiazoline ring, originally isolated from a secondary metabolite of the human commensal Staphylococcus lugdunensis (S. lugdunensis) [Zipperer A, et al. Nature 2016]. This groundbreaking discovery became a ray of hope in the search for new antibiotics, especially against difficult-to-treat drug-resistant strains such as MRSA (methicillin-resistant Staphylococcus aureus) and VRE (vancomycin-resistant Enterococci). Since its introduction in Nature in 2016, Lugdunin has attracted great interest due to its novel structure and strong antibacterial properties. However, the excitement about it has been somewhat dampened by the fact that its cyclopeptide composition includes alternating D / L hydrophobic amino acids that are incompatible with polar residues, which limits its modification potential. The thiazoline ring is key to its antibacterial effect, which further complicates structural modifications.

[0004] Although cationic antimicrobial peptides (CAMPs) are attractive for their ability to rapidly neutralize bacteria by disrupting membrane integrity, efforts to enhance Lugdunin by introducing cationic properties have been hampered by its intolerance to polar groups [Schilling NA, et al. Angew Chem Int Ed Engl 2019]. To date, no studies have successfully integrated polar amino acids into Lugdunin to optimize its physicochemical and antimicrobial properties, and no structures containing cationic amino acids and derivatives have been involved in existing patents on Lugdunin [Krismer B, et al. Patent EP3072899B1, US2018 / 0155397A1, and WO2016 / 151005AI].

[0005] In addition, patent document CN116635400A discloses a similar cyclic peptide compound composed of 6 amino acids or amino acid derivatives and a thiazolidine ring. The cyclic compound of the invention preferably has antimicrobial efficacy when used in cosmetics, especially antiviral, antibacterial and / or antifungal efficacy, preferably against Gram-positive bacteria. On the other hand, the cyclic compound of the invention, like Lugdunin, is completely composed of hydrophobic amino acids or amino acid derivatives, and does not contain any polar amino acids or amino acid derivatives.

[0006] Considering the natural source of Lugdunin and the inherent drug resistance of Staphylococcus Lyonnais, lugdunin faces the possibility of bacterial resistance in clinical treatment. Therefore, based on the skeleton structure of Lugdunin, a new type of cyclic peptide antibiotic containing cationic groups and a cationic antimicrobial peptide membrane-destructing action mode, which is easy to synthesize, will have better clinical application potential for resisting drug-resistant bacterial infections. Summary of the invention

[0007] The object of the present invention is to overcome at least one disadvantage of the prior art and provide a novel cationic cyclic peptide antibiotic based on Lugdunin.

[0008] The technical solution adopted by the present invention is:

[0009] In the first aspect, the present invention provides a novel cationic cyclic peptide antibiotic based on Lugdunin, wherein the general structural formula of the cationic cyclic peptide antibiotic is as shown in Formula I:

[0010]

[0011] At least one of the R1, R3, and R4 groups is selected from any one of the following cationic groups:

[0012] The other groups of the R1, R3, and R4 groups except the selected cationic group are selected from any one of the following structures:

[0013]

[0014] The R2 group is selected from any one of the following structures:

[0015] Wherein, m=0-6, n=0-6, p=1-4, q=1 or 2, and m+n=3-6; o=1 or 2;

[0016] And the salt, solvate, stereoisomer, tautomer or complex of the cationic cyclic peptide antibiotic shown in formula I.

[0017] In some examples, in the cationic cyclic peptide antibiotic, m=1; and / or n=2-4.

[0018] In some examples, the cationic cyclic peptide antibiotic is selected from any one of the following cyclic peptide antibiotics:

[0019]

[0020]

[0021]

[0022] In some embodiments, the amino acids in the cationic cyclic peptide antibiotic appear alternately in L- and D-forms.

[0023] In some examples, the cationic cyclic peptide antibiotic is selected from any one of the following cyclic peptide antibiotics:

[0024]

[0025]

[0026] In a second aspect, the present invention provides a method for preparing a medicament for preventing and treating drug-resistant bacterial infection by using the cationic cyclic peptide antibiotic provided in the first aspect of the present invention.

[0027] In some examples, the drug-resistant bacteria are selected from Gram-positive cocci, such as Staphylococci (including Staphylococcus aureus and Staphylococcus epidermidis) and Streptococci (such as Streptococcus agalactiae, Enterococcus, Streptococcus pneumoniae and Streptococcus pyogenes), Gram-positive bacilli, such as Bacillus anthracis, Listeria monocytogenes and Corynebacterium diphtheriae; Gram-negative cocci (such as Neisseria gonorrhoeae) and Gram-negative bacilli, such as Enterobacteriaceae (taking Escherichia coli as an example), Haemophilus influenzae, Citrobacter (such as friendly Citrobacter, Citrobacter multivariate), Salmonella and Shigella; as well as Klebsiella (such as Klebsiella pneumoniae and oxygenase Klebsiella), Enterobacteriaceae (such as Aeromonas and Aggregatimonas), Hafnia, Enterobacter sutschlandii (such as Enterobacter prausnitzii), Proteus (such as Proteus, Ritteri and Proteus), Providencia, Yersinia, and trophic Gram-negative rods. In addition, the antibacterial spectrum also includes Pseudomonas aeruginosa (such as Pseudomonas aeruginosa and Meketomonas) and strict anaerobes such as Clostridium fragilis, Peptidomonas, anaerobic Streptococci and Clostridium; as well as mycoplasmas (such as Mycoplasma pneumoniae, Mycoplasma hominis and Mycoplasma urogenitali), and mycobacteria such as Mycobacterium tuberculosis.

[0028] In some examples, diseases caused by the above-mentioned drug-resistant bacteria or mixed infections can be cured, prevented or alleviated by the compounds of the present invention, for example: human infectious diseases, such as septic infection, bone and joint infection, skin infection, postoperative wound infection, abscess, cellulitis, wound infection, infected burn, burn, oral infection, infection after dental surgery, septic arthritis, mastitis, tonsillitis, urogenital tract infection and eye infection.

[0029] In a third aspect, the present invention provides a drug for treating bacterial infection, wherein the active ingredient of the drug includes the cyclic peptide antibiotic described in the first aspect.

[0030] In some examples, it is used to treat, ameliorate and / or prevent bacterial infections.

[0031] In some examples, the bacteria is selected from the group consisting of Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci and Gram-negative bacilli, Klebsiella, Enterobacter, Hafnia, Steinernema, Proteus, Yersinia, and Eutropha.

[0032] The drug for treating bacterial infection provided by the present invention can be used not only to treat or prevent bacterial infection in humans, but also to treat in animals or plants. Examples are as follows:

[0033] Pigs: Escherichia coli diarrhea, enterotoxemia, sepsis, dysentery, salmonellosis, endometritis-mastitis-agalacia syndrome, mastitis;

[0034] Herbivores (cattle, sheep, goats): diarrhea, sepsis, bronchopneumonia, salmonellosis, pasteurellosis, mycoplasmosis, and reproductive tract infections;

[0035] Horses: bronchopneumonia, puerperal and postpartum infections, salmonellosis;

[0036] Dogs and cats: bronchopneumonia, diarrhea, dermatitis, otitis, urinary tract infection, prostatitis;

[0037] Poultry (chickens, turkeys, quail, pigeons, ornamental birds, etc.): Mycoplasmosis, Escherichia coli infection, chronic airway disease, Salmonellosis, Pasteurellosis, Psittacosis.

[0038] Similarly, bacterial diseases can be treated in the breeding and keeping of economic and ornamental fish. In addition, for humans, the antimicrobial spectrum of the mentioned pathogens is extended to pathogens such as Pasteurella, Brucella, Campylobacter, Listeria, Erysipelothrix, Corynebacterium, Borelia, Spirochetes, Nocardia, Rickettsia, Yersinia, etc.

[0039] Medicine for the treatment of bacterial infection provided by the invention can be used systemically and / or locally. For this reason, it can be used in a suitable manner, such as oral, parenteral, pulmonary, nasal, sublingual, tongue, cheek, rectum, skin, transdermal, conjunctiva, ear or in combination with implant or support.

[0040] The beneficial effects of the present invention are:

[0041] The cyclic peptide antibiotic of the present invention has good antibacterial activity, can cause cell membrane depolarization, destroy cell membrane integrity, quickly kill bacteria, inhibit the formation of biofilm, and is not easy to induce bacteria to produce drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a test of the hemolysis rate of mouse red blood cells at a concentration of 100 μg / mL in Example 4.

[0043] Figure 2 These are the bactericidal kinetic curves of cyclopeptide antibiotics 7, 41, 48, 53, 55, 60, 61, 67, 70, and 76 in Example 5.

[0044] Figure 3 Cyclic peptide antibiotics 7, 41, 48, 53, 55, 60, 61, 67, 70, and 76 induced drug resistance in Staphylococcus aureus, and the clinical antibiotic Amoxicillin was used as a positive control.

[0045] Figure 4The figures show the effects of cyclic peptide antibiotics 7 and 41 on the cell membrane potential of Staphylococcus aureus at concentrations of 1×MIC, 2×MIC, 4×MIC and 8×MIC; PBS was used as a negative control and 0.1% Triton X-100 was used as a positive control.

[0046] Figure 5 Flow cytometry was used to detect the changes in fluorescence intensity of Staphylococcus aureus and Escherichia coli after PI absorption induced by cyclopeptide antibiotic 41; PBS was used as a negative control.

[0047] Figure 6 Fluorescence photos of Staphylococcus aureus and Escherichia coli after incubation with a mixture of PI and Hoechst 33342 to detect the effect of cyclic peptide antibiotic 41 on Staphylococcus aureus and Escherichia coli using laser confocal microscopy; PBS was used as a negative control.

[0048] Figure 7 The inhibition rates of cyclic peptide antibiotics 7 and 41 on the biofilm formation of Staphylococcus aureus at concentrations of 1×MIC, 2×MIC and 4×MIC, and their clearance rates on the biofilm formed by Staphylococcus aureus at concentrations of 1×MIC, 2×MIC, 4×MIC, 8×MIC and 16×MIC.

[0049] Figure 8 Results of corneal colony counts and H&E staining of corneal tissue sections after treatment of MRSA-infected mouse keratitis with cyclopeptide antibiotics 7 and 41.

[0050] Fig. 9 Figure 3. The number of colonies in the lungs of mice and the H&E staining results of lung tissue sections after the cyclopeptide antibiotics 7 and 41 were used to treat pneumonia in mice infected with MRSA. Vancomycin was used as a positive control.

[0051] Fig.10 This is a test of the antifouling ability of cyclic peptide antibiotic 11 against Staphylococcus aureus after it was grafted onto the surface of contact lens. DETAILED DESCRIPTION

[0052] The following disclosure provides many different embodiments or examples for implementing different solutions of the present invention.

[0053] The embodiments of the present invention are described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0054] The instruments used in the present invention include: an orbital decolorization shaker (SK-O330, Dalong Xingchuang Experimental Instrument (Beijing) Co., Ltd., China), a rotary evaporator (560-00000-00-0, Heidolph, Germany), a time-of-flight mass spectrometer (Maxis 4G, Bruker, Germany), a HPLC preparative chromatogram (NU3001, Hanbang Technology, China), an analytical chromatogram (A02996, Waters, USA), a freeze dryer (CoolSafe55-4, Gene Company Limited, China), constant temperature shaker (IS-RDD3, Suzhou Jiemei Electronics Co., Ltd., China), biological safety cabinet (BSC-1500ⅡB2-X, Jinan Xinbeisi Biotechnology Co., Ltd., China), constant temperature incubator (LT-BIX300M, Lidetai (Shanghai) Scientific Instrument Co., Ltd.), desktop centrifuge (Thermo75004250, Thermo Fisher Scientific Inc., USA), multifunctional microplate reader (FlexStation III, Meigu Molecular Instruments, USA), carbon dioxide cell culture incubator (CLM-170B-8-TC, Taicang Yisigao Medical Equipment Technology Co., Ltd., China), fluorescence microscope (U-LH50HG, Olympus Corporation, Japan), micro-nucleic acid protein analyzer (E113192, Implen GmbH, Germany), mouse laryngoscope and nebulizer (Yuansen Kaide Biotechnology Co., Ltd., China), tissue cryo-grinding instrument (JXFSPRP-CLN-48, Shanghai Jingxin Industrial Development Co., Ltd., China), X-ray photoelectron spectroscopy (XPS) (Kratos AXIS Ultra DLD, UK), flow cytometer (NovoCyte Quanteon, Agilent)

[0055] The reagents used were: o-benzotriazole-N, N, N, N'-tetramethyl-hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBT), N, N-diisopropylethylamine (DIPEA), trifluoroacetic acid (TFA) and dichloromethane (DCM) were purchased from Xiens Technology Co., Ltd. (China). N, N-dimethylformamide (DMF) was provided by BASF (Germany). Dess-Martin periodinane, N, N'-carbonyldiimidazole and sodium borohydride were provided by Myril Technology Co., Ltd. Rink Amide MBHA resin was provided by Lanxiao Technology (Xi'an, China). Acetonitrile was provided by Shanghai CINC High Purity Solvent Co., Ltd. (Shanghai, China). D, L-amino acids and cyclophosphamide were purchased from Myril Chemical Technology Co., Ltd. Ether, piperidine and hydrochloric acid were purchased from Shantou Xilong Chemical Co., Ltd. (Shantou, China). Dimethyl sulfoxide (DMSO) and 5,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Tianjin Guangfu Reagent Co., Ltd. MH broth medium was purchased from Qingdao Hi-Tech Park Haibo Biotechnology Co., Ltd., and PBS buffer was purchased from Wuhan Saiweier Biotechnology Co., Ltd. TritonX-100 was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. RPMIDMEM medium was purchased from Shanghai Dathill Biotechnology Co., Ltd. Fetal bovine serum (FBS) was purchased from Shanghai Dathill Biotechnology Co., Ltd. Trypsin cell digestion solution was purchased from Beijing Lanjieke Technology Co., Ltd. Agar powder was purchased from Beijing Lanjieke Technology Co., Ltd.

[0056] Example 1 Synthesis of cyclic peptide antibiotics

[0057] The synthesis of cyclic peptide antibiotics is divided into two steps: aldehyde resin synthesis and peptide synthesis:

[0058] 1.1 Synthesis of formaldehyde resin (taking amic acid as an example):

[0059] First, Fmoc-valine is used as a raw material to synthesize Fmoc-valinaldehyde, which is then reacted with serine to generate 2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-methylpropyl)oxazolidine-4-carboxylic acid. The compound is then protected with BOC anhydride and finally connected with MBHA resin through an amide condensation reaction to generate an aldehyde-producing resin.

[0060] 1.2 Peptide synthesis:

[0061] There are two strategies for peptide synthesis. The first strategy is to first follow the classic Fmoc solid-phase peptide synthesis method to gradually connect the Fmoc-protected amino acids to the synthesized aldehyde resin in the order of the peptide chain to synthesize a linear peptide chain; then, use a cleavage reagent (prepared in the following ratios: TFA: DCM: TIS: H2O = (1-30): (70-99): (0-5): (0-5) to remove the side chain protection to release the linear peptide aldehyde from the resin, and then the C-terminal aldehyde of the peptide aldehyde can undergo an intramolecular cyclization reaction with the N-terminal cysteine ​​to form a thiazolidine ring, thereby forming a cyclic peptide. The second strategy is to first follow the classic Fmoc solid-phase peptide synthesis method to gradually connect the Fmoc-protected amino acids to the synthesized aldehyde resin in the order of the peptide chain to synthesize a linear peptide chain; then, use a cleavage reagent (prepared in the following ratios: TFA: DCM: TIS: H2O = (1-30): (70-99): (0-5): (0-5) to remove the side chain protection to release the linear peptide aldehyde from the resin, and then the C-terminal aldehyde of the peptide aldehyde can undergo an intramolecular cyclization reaction with the N-terminal cysteine ​​to form a thiazolidine ring, thereby forming a cyclic peptide. The peptide chains are connected step by step to 2-chlorotrityl chloride resin in sequence, and then, a cleavage agent (TFE:DCM=30:70) is used to cleave the polypeptide from the resin, and the polypeptide obtained by cleavage is added to DMF, and condensation agents HOBT, HBTU and DIEA are added to cyclize the head and tail amides of the polypeptide. After the cyclization is completed, it is washed with ethyl acetate and water, and the organic phase is collected. The organic phase is removed by a rotary evaporator to obtain a white solid, and a cleavage agent (TFA:DCM:TIS:H2O prepared in the following ratio=(1-30):(70-99):(0-5):(0-5)) is added to remove the side chain protection to obtain a cyclic peptide antibiotic.

[0062] Example 2 Purification and Preparation of Cyclic Peptide Antibiotics

[0063] The synthesized cyclopeptide antibiotics were separated and purified by high performance liquid chromatography. Simply, the synthesized cyclopeptide crude sample was washed and precipitated with ether, and the remaining ether was removed by a rotary evaporator, and then the precipitate was dissolved and purified by gradient elution using an acetonitrile (0.1% TFA) / water (0.1% TFA) system. The purified product was identified by mass spectrometry and freeze-dried to obtain the cyclopeptide antibiotic.

[0064] Example 3 Determination of in vitro antibacterial activity of cyclic peptide antibiotics

[0065] Cyclic peptide antibiotics 1 to 77 were synthesized according to the process described in Example 1 and purified according to the purification method described in Example 2. The strains used in the determination of the in vitro antibacterial activity of the cyclic peptide antibiotics and the control antibiotics include standard strains (including Escherichia coli (E. coli ATCC 25922, C. albicans ATCC 14053), Staphylococcus aureus (S. aureus ATCC 25923 、MRSA ATCC 43300), Bacillus subtilis (B. subtilis ATCC 23875), Staphylococcus epidermidis (S. epidermidis ATCC 12228), Enterococcus faecalis (E. faecalis ATCC 19433, VRE ATCC 51299), and Enterococcus gallinarum (E. gallinarum ATCC 49573), all from the American Type Culture Collection) and clinically isolated drug-resistant strains (including MSRA S2, MSRA S3, MSRA S4, provided by the First Hospital of Lanzhou University).

[0066] The minimum inhibitory concentration (MIC) values of the antibiotics and control antibiotics against the tested strains were determined using the standard two-fold dilution method recommended by the National Committee for Clinical and Laboratory Standards (NCCLS) of the United States. Briefly, an appropriate amount of bacterial (or fungal) frozen stock solution was added to fresh MH / SD medium, and the bacterial solution was placed on a shaker at 37°C and cultured overnight at 180 rpm. The above bacterial solution was sub-cultured twice and continued to be cultured on the shaker for 4 - 5 hours to obtain bacteria in the logarithmic growth phase. Then, 1×10 6 CFU / mL of the bacterial solution was added to a 96-well plate, 100 μL per well. Then, 100 μL of different concentrations of the polypeptide (1 - 200 μg / mL) twice the final concentration was added to each well in a two-fold dilution manner. The negative control was fresh culture medium, and three parallels were made for each concentration. After adding the drugs, the 96-well plate was placed in a constant temperature and humidity incubator at 37°C for 18 - 24 hours, and then the results were observed. The concentration of the cyclic peptide antibiotic at the first clear well after the visibly turbid well was recorded as the minimum inhibitory concentration of the cyclic peptide antibiotic against the bacterium, that is, its MIC value for the bacterium.

[0067] In this study, vancomycin and linezolid were used as controls. The antibacterial activities of cyclic peptide antibiotics 1 - 77 against the tested bacteria are shown in Table 1, where "++++" indicates MIC ≤ 12.5 μg / mL, "+++" indicates 12.5 μg / mL < MIC ≤ 25 μg / mL, "++" indicates 25 μg / mL < MIC ≤ 50 μg / mL, "+" indicates 50 μg / mL < MIC ≤ 100 μg / mL, and "-" indicates MIC > 100 μg / mL.

[0068] Table 1

[0069]

[0070]

[0071]

[0072] Example 4 Determination of hemolytic activity of cyclic peptide antibiotics 7, 9, 24, 25, 31, 37, 41, 43, 46, 48, 53, 55, 60, 61, 67, 70 and 76 on mammalian erythrocytes (HC 50 >100 μg / mL)

[0073] The OD of the red blood cell solution was measured by using a microplate reader after the red blood cells were treated with cyclopeptide antibiotics 7, 9, 24, 25, 31, 37, 41, 43, 46, 48, 53, 55, 60, 61, 67, 70 and 76. 490 The hemolytic activity of cyclopeptide antibiotics was evaluated by using the values ​​obtained from the ischemic centrifugation of mouse eyeballs.

[0074] First, the whole blood of the mice was centrifuged at 1200rpm for 5min and the supernatant was discarded. After washing with PBS, 100% of the red blood cells were collected and diluted to 8% and added to a 96-well plate. The concentration of the cyclic peptide antibiotic to be tested was 100μg / mL and then incubated in a cell culture incubator. After 1h, the 96-well plate was centrifuged at 1500rpm for 15min, and then the supernatant was transferred to another 96-well plate and the OD was measured with an enzyme marker. 490 Finally, according to the formula: hemolysis rate (%) = (experimental group OD 490 - Negative control group OD 490 ) / (OD of positive control group 490 - Negative control group OD 490 )×100% to calculate the hemolytic rate of each cyclic peptide antibiotic.

[0075] like Figure 1 As shown, the hemolytic rate of cyclic peptide antibiotic 9 exceeds 20%, and the other cyclic peptide antibiotics are weakly toxic to red blood cells. The hemolytic rates of cyclic peptide antibiotics 24, 25, and 37 are between 10% and 20%, and the hemolytic toxicity of cyclic peptide antibiotics 7, 31, 41, 43, 46, 48, 53, 55, 60, 61, 67, 70, and 76 are between 1% and 10%.

[0076] Example 6 Determination of bactericidal kinetics of cyclic peptide antibiotics

[0077] Cyclic peptide antibiotics 7, 41, 48, 53, 55, 60, 61, 67, 70, and 76 were used as representatives to determine the effect of cyclic peptide antibiotics on the growth and proliferation of Staphylococcus aureus ATCC 25923 at different time intervals by colony count (CFU) counting method. Then, the time interval was used as the horizontal axis and the logarithmic value of the number of colonies per milliliter was used as the vertical axis to plot the bactericidal kinetics of cyclic peptide antibiotics. Simply put, the logarithmic concentration was 1.0×10 6CFU / mL of Staphylococcus aureus was added to a 96-well plate, and then cyclotide antibiotics (final concentration of 4×MIC) were added respectively, and co-cultured at 37°C. At 0h, 1 / 6h, 1 / 2h, 1h, 2h, 4h, 8h, 12h and other time intervals, appropriate amounts of bacterial solution were taken out and diluted to an appropriate multiple, and 100 μL of the diluted bacterial solution was evenly spread on MH solid culture medium with a spreading rod, and the colonies were counted after overnight culture.

[0078] Figure 2 It showed that at a concentration of 4×MIC, cyclopeptide antibiotics 7, 48, 55, 61, 67 and 76 could kill bacteria within 2 hours, and cyclopeptide antibiotics 41, 53, 60 and 70 could kill bacteria within 4 hours, all of which showed the ability to kill bacteria rapidly.

[0079] Example 7. Cyclic peptide antibiotic resistance assay

[0080] The potential of cyclic peptide antibiotics to induce bacterial resistance was evaluated by measuring the minimum inhibitory concentration of cyclic peptide antibiotics against Staphylococcus aureus ATCC 25923 for 21 consecutive times. Simply, Staphylococcus aureus in the logarithmic growth phase was diluted and added to a 96-well microtiter plate, and its MIC was determined using the classic two-fold dilution method. The concentration of the first well where no turbidity was observed by the naked eye was recorded as the initial MIC0. Then, the bacterial solution in 1 / 2×MIC0 well was used as the bacterial solution for the next MIC determination, and the first MIC was determined in the same way and recorded as MIC1. Next, the MIC value of the cyclic peptide antibiotic against Staphylococcus aureus was continuously determined in the same way, and the determination was continued for 21 times, which was recorded as MIC 21 Finally, a graph was drawn with the number of measurements as the horizontal axis and the ratio of the MIC value to the MIC0 value as the vertical axis to evaluate the occurrence of bacterial resistance induced by cyclopeptide antibiotics. Amoxicillin, a clinical antibiotic, was used as a control.

[0081] like Figure 3 As shown, in the 21-day bacterial resistance experiment, the MIC value of cyclopeptide antibiotic 7 was 3.125 μg / mL, the MIC value of cyclopeptide antibiotic 41 was 3.125 μg / mL, the MIC value of cyclopeptide antibiotic 48 was 6.25 μg / mL, the MIC value of cyclopeptide antibiotic 53 was 6.25 μg / mL, the MIC value of cyclopeptide antibiotic 55 was 3.125 μg / mL, the MIC value of cyclopeptide antibiotic 60 was 6.25 μg / mL, the MIC value of cyclopeptide antibiotic 61 was 6.25 μg / mL, the MIC value of cyclopeptide antibiotic 67 was 6.25 μg / mL, the MIC value of cyclopeptide antibiotic 70 was 6.25 μg / mL, and the MIC value of cyclopeptide antibiotic 76 was 12.5 μg / mL, which was 1-2 times higher than the initial MIC. The bacteria did not develop resistance to it.

[0082] The initial MIC of the amoxicillin group was 0.16 μg / mL, the MIC value on day 7 was 8 times the initial value, the MIC value on day 12 was 32 times the initial value, and the MIC value on day 16 was 250 times the initial value.

[0083] Example 8. Effects of cyclic peptide antibiotics on bacterial cell membranes

[0084] The effect of cyclic peptide antibiotics on bacterial cell membrane was evaluated by detecting the disruptive effect of cyclic peptide antibiotics represented by 7 and 41 on the cell membrane potential of Staphylococcus aureus ATCC 25923 and the PI absorption effect caused by Staphylococcus aureus.

[0085] 8.1 Disruption of Staphylococcus aureus Membrane Potential by Cyclic Peptide Antibiotics

[0086] The disruptive effect of cyclopeptide antibiotics on the membrane potential of Staphylococcus aureus was determined by the DiOC2(3) fluorescent probe method. Briefly, Staphylococcus aureus ATCC 25923 was incubated at 37°C for 18 hours and centrifuged at 5000 rpm for 10 minutes. The precipitate was collected and washed three times with 10 mM PBS (pH 7.4) containing 0.1% glucose and then resuspended to OD 600 =0.5. Subsequently, an equal volume of 60 μM DiOC2(3) solution was added to the bacterial suspension and incubated at 37°C in the dark for 15 minutes. Then, the bacterial suspension containing DiOC2(3) (50 μL / well) was added to a 96-well microtiter plate, followed by the addition of 50 μL of different concentrations of cyclopeptide antibiotics (ranging from 1 to 8 times the MIC) and incubation for 30 minutes. Finally, an ELISA reader (Synergy NEO2, Agilent) was used to detect the fluorescence of each well, with an excitation wavelength of 485 nm and two emission wavelengths of 530 nm and 630 nm, respectively. 0.1% Triton X-100 was used as a positive control and PBS was used as a negative control.

[0087] like Figure 4 As shown, cyclic peptide antibiotics 7 and 41 disrupted the bacterial membrane potential in a concentration-dependent manner and were able to completely disrupt the bacterial membrane potential at concentrations of 4×MIC and 8×MIC.

[0088] 8.2 Determination of the effects of cyclic peptide antibiotics on the destruction of bacterial cell membrane integrity.

[0089] The disruptive effect of cyclopeptide antibiotics on bacterial cell membrane integrity was evaluated by measuring the absorption of the fluorescent dye propidium iodide (PI) by Staphylococcus aureus (ATCC 25923) after the action of cyclopeptide antibiotics. Briefly, after overnight incubation of Staphylococcus aureus at 37°C, the bacterial solution was centrifuged at 5000 rpm for 10 minutes, washed three times with PBS (10 mM, pH = 7.4), and suspended to 1×10 9 CFU / mL concentration. Subsequently, 100 μL of S. aureus suspension was thoroughly mixed with an equal volume of cyclotide antibiotic solution and incubated in a shaker at 37 °C for 1 hour. Then, 10 μL of PI (1 mg / mL) was added and incubated in the dark for 15 minutes for flow cytometer (NovoCyte Quanteon, Agilent) analysis. The untreated group served as a negative control; alternatively, 10 μL each of PI (10 μM) and Hoechst 33342 (10 μM) was added and incubated in the dark for 15 minutes for fluorescence inverted microscope photography.

[0090] like Figure 5 As shown, after Staphylococcus aureus was treated with cyclopeptide antibiotic 41 at a concentration of 4×MIC, the positive rate of PI detected by flow cytometry was 98.27%, while the positive rate of PI in Staphylococcus aureus treated with PBS was only 10.66%. The positive rate of PI in Escherichia coli treated with cyclopeptide antibiotic 41 at a concentration of 4×MIC was 74.68%, while the positive rate of PI in Escherichia coli treated with PBS was 11.82%. This shows that cyclopeptide antibiotic 41 can destroy the cell membrane of bacteria.

[0091] like Figure 6 As shown, obvious red fluorescence can be observed in Staphylococcus aureus treated with cyclic peptide antibiotic 41, while no red fluorescence is detected in Staphylococcus aureus treated with PBS. Obvious red fluorescence can be observed in Escherichia coli treated with cyclic peptide antibiotic 41, while no red fluorescence is detected in Escherichia coli treated with PBS. This indicates that cyclic peptide antibiotic 41 can destroy the bacterial cell membrane, causing PI to enter the bacteria.

[0092] Example 9. Activity determination of cyclic peptide antibiotics in inhibiting biofilm formation of Staphylococcus aureus

[0093] The inhibition of biofilm formation of Staphylococcus aureus (ATCC 25923) by cyclic peptide antibiotics was determined using the crystal violet method. Briefly, a concentration of 1.0×10 6CFU / mL of logarithmic phase Staphylococcus aureus was added to a 96-well plate, and then the cyclopeptide antibiotics 7 and 41 to be tested were added at a concentration of 1×MIC-16×MIC gradient concentrations, and incubated in a 37°C constant temperature bacterial incubator. After 24 hours, the 96-well plate was removed to remove the planktonic bacteria on the surface, washed with PBS, fixed with methanol, and stained with crystal violet solution for 15 minutes, and then 95% ethanol was added to dissolve the crystal violet in the biofilm to measure the OD 570 According to the formula, biofilm formation inhibition rate (%) = [1-(experimental group OD 570 - Negative control group OD 570 ) / (OD of positive control group 570 - Negative control group OD 570 )]×100%.

[0094] like Figure 7 As shown, cyclopeptide antibiotic 7 can inhibit more than 95% of Staphylococcus aureus biofilm formation at a concentration of 1×MIC, cyclopeptide antibiotic 41 can inhibit 50% of Staphylococcus aureus biofilm formation at a concentration of 1×MIC, and can inhibit more than 95% of Staphylococcus aureus biofilm formation at a concentration of 2×MIC. Cyclic peptide antibiotic 7 can eliminate 40% of the already formed Staphylococcus aureus biofilm at a concentration of 4×MIC, and can eliminate 90% of the already formed Staphylococcus aureus biofilm at a concentration of 8×MIC. Cyclic peptide antibiotic 41 can eliminate 50% of the already formed Staphylococcus aureus biofilm at a concentration of 4×MIC, and can eliminate 85% of the already formed Staphylococcus aureus biofilm at a concentration of 8×MIC.

[0095] Example 9. Determination of antibacterial activity of cyclic peptide antibiotics in vivo

[0096] The in vivo antibacterial activity of cyclic peptide antibiotics was tested, represented by cyclic peptide antibiotics 7 and 41. The in vivo antibacterial activity of cyclic peptide antibiotics was evaluated by establishing a mouse keratitis model and a mouse pneumonia model infected with MRSA and testing their therapeutic effects in these animal models.

[0097] 9.1 Mouse keratitis model

[0098] When testing the in vivo antibacterial activity of cyclopeptide antibiotics in the mouse keratitis model, Kunming mice were randomly divided into a negative control group, a cyclopeptide antibiotic 7 model group, a cyclopeptide antibiotic 41 model group, a cyclopeptide antibiotic 7 treatment group, a cyclopeptide antibiotic 41 treatment group, a linezolid control group, and a vancomycin control group. After anesthesia, the mice in the model group, the treatment group, the linezolid control group, and the vancomycin control group were anesthetized, and the cornea of ​​the mice was scratched with a needle, and then infected with 10 μL of MRSA bacterial suspension to establish a keratitis model. 12 hours after infection, the mice in the cyclopeptide antibiotic 7 treatment group and the linezolid control group were treated with 10 μL of 1 mg / mL cyclopeptide antibiotic 7 and 1 mg / mL linezolid for multiple eye drops. The cyclopeptide antibiotic 41 treatment group and the vancomycin control group were treated with 10 uL of 1 mg / L cyclopeptide antibiotic 41 and 1 mg / mL vancomycin for multiple eye drops. In contrast, the model group received 10 μL of 0.9% saline each time. After 8 hours of treatment, the mice were sacrificed and their ocular tissues were collected for bacterial colony counts and H&E staining histological examination.

[0099] like Figure 8 The results showed that the colony count of the eyeball of mice treated with cyclopeptide antibiotic 7 (1 mg / ml) was reduced by 0.854 log (p<0.05) compared with the model group, which was equivalent to the group treated with linezolid (1 mg / ml) (reduced by 0.864 log, p<0.05). The results of H&E staining showed that the cornea of ​​the model group was defective and a large number of inflammatory cells were gathered, while the corneal epithelial cells and basal layer of the cyclopeptide antibiotic 7 and linezolid treatment groups were relatively intact, with fewer inflammatory cells. The colony count of the eyeball of mice treated with cyclopeptide antibiotic 41 (1 mg / ml) was reduced by 1.244 log (p<0.05) compared with the model group, which was equivalent to the group treated with vancomycin (1 mg / ml) (reduced by 1.305 log, p<0.05). The results of H&E staining showed that the corneal surface of the model group was rough, with a large number of inflammatory cells gathered, while the corneal epithelial cells and basal layer of the cyclopeptide antibiotic 41 and vancomycin treatment groups were relatively smooth, with fewer inflammatory cells.

[0100] 9.2 Mouse pneumonia model

[0101] To determine the therapeutic potential of cyclopeptide antibiotics in a mouse pneumonia model, female Kunming mice were randomly divided into an untreated control group, a model group, a cyclopeptide antibiotic 7 treatment group, a cyclopeptide antibiotic 41 treatment group, and a vancomycin control group. After anesthetization of the mice in the model group, cyclopeptide antibiotic treatment group 1, cyclopeptide antibiotic treatment group 2, and vancomycin control group, 25 μL of OD 600A 0.35 MRSA (ATCC 43300) bacterial suspension was injected into the mouse trachea. At 2 hours and 12 hours after infection, mice in the model group, cyclopeptide antibiotic 7 treatment group, cyclopeptide antibiotic 41 treatment group and vancomycin control group were treated with intraperitoneal injection of normal saline, cyclopeptide antibiotics 7 and 41 (5 mg / kg) and vancomycin (5 mg / kg), respectively. 24 hours after infection, all mice were killed, and bacterial colony counts and H&E staining histological examinations were collected.

[0102] like Fig. 9 As shown, the cyclotide antibiotic 7 treatment group (5 mg / kg) can effectively reduce the lung bacterial load within 24 hours (reduced by 0.56 log, p<0.05), which is comparable to the vancomycin (5 mg / kg) treatment group (reduced by 0.76 log, p<0.05). The results of H&E staining showed that the alveolar morphology of the cyclotide antibiotic 7 treatment group was intact, and no obvious inflammatory cells and capillary congestion were observed, which was similar to the lung characteristics of the vancomycin group and the uninfected group, while the alveolar capillaries in the model group were congested, and an increase in inflammatory cells could be observed. The cyclotide antibiotic 41 treatment group (5 mg / kg) can effectively reduce the lung bacterial load within 24 hours (reduced by 0.62 log, p<0.05), which is comparable to the vancomycin (5 mg / kg) treatment group (reduced by 0.54 log, p<0.05). The results of H&E staining showed that the alveolar morphology of the group treated with cyclopeptide antibiotic 41 was intact, and no obvious inflammatory cells and capillary congestion were observed, which was similar to the lung characteristics of the vancomycin group and the uninfected group. In the model group, the alveolar wall was thickened, the alveolar tissue was reduced, the capillaries were congested, and an increase in inflammatory cells could be observed.

[0103] Example 10. Determination of the antifouling effect of cyclic peptide antibiotics grafted onto the surface of contact lenses

[0104] Contact lenses were used as implant representatives to evaluate the antimicrobial potential of cyclic peptide antibiotics after grafting onto the implant surface. First, (3-aminopropyl) trimethoxysilane was linked to the hydroxyl groups on the surface of the contact lens. Then, 6-azidohexanoic acid was attached to (3-aminopropyl) trimethoxysilane via an amide condensation reaction. Subsequently, the alkynyl group of the cyclic peptide antibiotic 11 was linked to the azide group on the surface of the contact lens via a click chemistry reaction, completing the grafting of the cyclic peptide antibiotic onto the surface of the contact lens. Characterization was performed using X-ray photoelectron spectroscopy (XPS) (Kratos AXIS Ultra DLD, UK). Finally, the antimicrobial effect was evaluated by incubating the contact lenses grafted with the cyclic peptide antibiotic 11 with a suspension of Staphylococcus aureus and evaluating the bacterial colony counts on the lens surface. Contact lenses without grafted cyclic peptide antibiotics were used as negative controls.

[0105] like Fig.10As shown, contact lenses grafted with cyclic peptide antibiotic 11 were able to reduce bacterial attachment by 63% (p<0.05) compared with the ungrafted group, demonstrating the great potential of this series of cyclic peptide antibiotics in preventing implant-related infections.

[0106] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A novel cationic cyclic peptide antibiotic based on Lugdunin, characterized in that: The general structural formula of the cationic cyclic peptide antibiotic is shown in Formula I: At least one of the R1, R3, and R4 groups is selected from any one of the following cationic groups: The other groups of the R1, R3, and R4 groups except the selected cationic group are selected from any one of the following structures: The R2 group is selected from any one of the following structures: Wherein, m=0-6, n=0-6, p=1-4, q=1 or 2, and m+n=3-6; o=1 or 2; And the salt, solvate, stereoisomer, tautomer or complex of the cationic cyclic peptide antibiotic shown in formula I.

2. The cationic cyclic peptide antibiotic according to claim 1, characterized in that In the cationic cyclic peptide antibiotic, m=1; And / or n=2-4.

3. The cationic cyclic peptide antibiotic according to claim 1, characterized in that The cationic cyclic peptide antibiotic is selected from any one of the following cyclic peptide antibiotics:

4. The cationic cyclic peptide antibiotic according to claim 3, characterized in that The amino acids in cationic cyclic peptide antibiotics appear alternately in L-, D-form or D-, L-form.

5. The cationic cyclic peptide antibiotic according to claim 1, characterized in that The cationic cyclic peptide antibiotic is selected from any one of the following cyclic peptide antibiotics:

6. Use of the cationic cyclic peptide antibiotic according to claim 1 in the preparation of a drug for resisting drug-resistant bacterial infection.

7. The use according to claim 6, characterized in that: The drug-resistant bacteria are selected from Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci and Gram-negative bacilli, Klebsiella, Enterobacter, Hafnia, Steinernema, Proteus, Yersinia, and atrophic Gram-negative bacilli.

8. A drug for treating bacterial infection, characterized in that: The active ingredient comprises the cyclic peptide antibiotic according to any one of claims 1 to 5.

9. The drug according to claim 8, characterized in that It is used to treat, improve and / or prevent bacterial infections.

10. The drug according to claim 8, characterized in that The bacteria are selected from the group consisting of Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci and Gram-negative bacilli, Klebsiella, Enterobacter, Hafnia, Steinernema, Proteus, Yersinia, and Eutrophic Gram-negative bacilli.

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