Novel cationic cyclic peptide antibiotics based on lugdunin

Abstract

The application discloses a cationic cyclic peptide antibiotic based on Lugdunin. The cationic cyclic peptide antibiotic provided by the application has the membrane damage 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 present invention is in the field of organic chemistry and relates specifically to novel cationic cyclic peptide antibiotics based on Lugdunin. BACKGROUND

[0002] With the increasing prevalence of drug-resistant bacterial infections, antibiotic resistance has emerged as a major challenge to human health and medical practice. Drug-resistant bacteria will make some common infectious diseases that have been easily treated with traditional antibiotics since the discovery of penicillin once again difficult to treat. In addition to increasing the risk of treatment failure and complications, drug-resistant bacterial infections can also lead to an increase in infection-related disease mortality. Currently, about 700,000 patients die from drug-resistant bacterial infections each year. If effective antimicrobial drugs are not discovered, this number will increase to 100 million per year by 2050, with a direct economic loss of over $10 billion. In addition, the widespread spread of drug-resistant bacteria will make modern medical procedures such as surgery and organ transplantation susceptible to infection, affecting the implementation of these procedures and even hindering the development of medical technology. However, due to the low commercial returns of current antimicrobial agents, companies lack enthusiasm and investment in researching antimicrobial agents, resulting in a severe shortage of antimicrobial agent development 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 cyclic peptide antibiotic containing a thiazoline ring, which was originally isolated from the secondary metabolites of the human commensal Staphylococcus lugdunensis (also known as S. lugdunensis) [Zipperer A, et al. Nature 2016]. This groundbreaking discovery has become a beacon of hope for the search for new antibiotics, especially for hard-to-tackle drug-resistant strains such as MRSA (methicillin-resistant Staphylococcus aureus) and VRE (vancomycin-resistant Enterococcus). Since its introduction in Nature in 2016, Lugdunin has generated great interest due to its novel structure and strong antimicrobial properties. However, the excitement has been tempered somewhat due to its cyclic peptide composition, which includes alternating D / L hydrophobic amino acids that are incompatible with polar residues, limiting its modification potential. The thiazoline ring is key to its antimicrobial effect, which further complicates structural changes.

[0004] Although cationic antimicrobial peptides (CAMPs) are attractive in rapidly neutralizing bacteria by disrupting membrane integrity, efforts to enhance Lugdunin by introducing cationic properties are hindered by its intolerance to polar groups [Schilling NA, et al. Angew Chem Int Ed Engl 2019]. To date, no study has successfully integrated polar amino acids into Lugdunin to optimize its physicochemical and antibacterial properties, and none of the existing patents on Lugdunin have involved any structures containing cationic amino acids and derivatives [Krismer B, et al. Patent EP3072899B1, US2018 / 0155397A1, and WO2016 / 151005AI].

[0005] In addition, patent document CN116635400A discloses a similar cyclic peptide compound consisting of 6 amino acids or amino acid derivatives and a thiazolidine ring. The cyclic compound of the invention preferably has antimicrobial efficacy, especially antiviral, antibacterial and / or antifungal efficacy, preferably against gram-positive bacteria, when used in cosmetics. However, on the other hand, the cyclic compound of the invention, like Lugdunin, is completely composed of hydrophobic amino acids or amino acid derivatives, without any polar amino acids or amino acid derivatives.

[0006] Considering the natural source of Lugdunin and the inherent drug resistance of Staphylococcus lugdunensis, lugdunin faces the possibility of bacterial drug resistance in clinical treatment. Therefore, developing a new class of cyclic peptide antibiotics with cationic groups based on the skeleton structure of Lugdunin, which has the membrane disruption mode of cationic antimicrobial peptides and is easy to synthesize, will have better potential for clinical application to resist drug-resistant bacterial infections. SUMMARY

[0007] The purpose of the present application is to overcome at least one deficiency of the prior art and provide a new cationic cyclic peptide antibiotic based on Lugdunin.

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

[0009] In a first aspect, the present application provides a new cationic cyclic peptide antibiotic based on Lugdunin, the structural general formula of the cationic cyclic peptide antibiotic is shown as formula I:

[0010]

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

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

[0013]

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

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

[0016] And salts, solvates, stereoisomers, tautomers, or complexes of the cationic cyclic peptide antibiotics shown in Formula I.

[0017] In some instances, the cationic cyclic peptide antibiotic has m = 1; and / or n = 2 to 4.

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

[0019]

[0020]

[0021]

[0022] In some instances, the amino acids in cationic cyclic peptide antibiotics alternate between L- and D-types.

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

[0024]

[0025]

[0026] Secondly, the use of the cationic cyclic peptide antibiotic provided in the first aspect of the present invention in the preparation of a medicament for treating drug-resistant bacterial infections.

[0027] In some instances, the drug-resistant bacteria are selected from Gram-positive cocci, such as Staphylococcus (including Staphylococcus aureus and Staphylococcus epidermidis) and Streptococcus (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 (e.g., Escherichia coli), Haemophilus influenzae, Citrobacter (such as Citrobacter zedoaria and Citrobacter polymorpha), Salmonella, and Shigella; as well as Klebsiella pneumoniae (such as Klebsiella pneumoniae and Klebsiella oxytetracycline), Enterobacteriaceae (such as Aeromonas and Polymonas), Hafnia, Enterobacter strychnifolia (such as Enterobacter plastriatus), Proteus (such as Proteus, Reiterella, and Proteus), Providencia, Yersinia, and trophotropic Gram-negative bacilli. In addition, the antibacterial spectrum includes Pseudomonas aeruginosa (such as Pseudomonas aeruginosa and Meckiae) and strict anaerobes, such as Clostridium fragilis, Peptococcus, anaerobic streptococci and Clostridium spp.; as well as mycoplasma (such as Mycoplasma pneumoniae, Mycoplasma hominis and Mycoplasma urogenitalia), and mycobacteria, such as Mycobacterium tuberculosis.

[0028] In some instances, diseases caused by the aforementioned drug-resistant bacteria or mixed infections can be cured, prevented, or alleviated by the compounds of the present invention, such as human infectious diseases, including septic infections, bone and joint infections, skin infections, postoperative wound infections, abscesses, cellulitis, wound infections, infectious burns, scalds, oral infections, post-dental infections, septic arthritis, mastitis, tonsillitis, urogenital infections, and eye infections.

[0029] Thirdly, the present invention provides a medicament for treating bacterial infections, the active ingredient of which includes the cyclic peptide antibiotic described in the first aspect.

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

[0031] In some instances, the bacteria are selected from Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci and Gram-negative bacilli, Klebsiella, Enterobacter, Hafnia, Enterobacter schoenleinii, Proteus, Yersinia, and trophotropic Gram-negative bacilli.

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

[0033] Pigs: E. coli diarrhea, enterotoxemia, sepsis, dysentery, salmonellosis, endometritis-mastitis-agalactia 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, quails, pigeons, ornamental birds, etc.): Mycoplasma infection, E. coli infection, chronic airway disease, salmonellosis, pasteurellosis, psittacosis.

[0038] Similarly, it can be used to treat bacterial diseases in the breeding and raising of economic and ornamental fish. In addition, for humans, the antibacterial spectrum of the mentioned pathogens has been extended to include pathogens such as Pasteurella, Brucella, Campylobacter, Listeria, Erysipelothrix rhusiopathiae, Corynebacterium, Borelia, Spirochetes, Nocardia, Rickettsia, Yersinia, etc.

[0039] The medicament for treating bacterial infections provided by this invention can exert systemic and / or local effects. Therefore, it can be administered in appropriate ways, such as orally, parenterally, pulmonaryly, nasally, sublingually, on the tongue, cheek, rectum, through the skin, transdermally, through the conjunctiva, intraauricularly, or in combination with implants or stents.

[0040] The beneficial effects of this invention are:

[0041] The cyclic peptide antibiotic of the present invention has good antibacterial activity. It can cause cell membrane depolarization, disrupt cell membrane integrity, kill bacteria rapidly, inhibit the formation of biofilms, and is not likely to induce bacterial resistance. Attached Figure Description

[0042] Figure 1 This is a test of the hemolysis rate of mouse erythrocytes at a concentration of 100 μg / mL, as described in Example 4.

[0043] Figure 2 The images show the bactericidal kinetic curves of the cyclic peptide 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 were used to induce drug resistance in Staphylococcus aureus, with the clinically used antibiotic Amoxicillin serving as a positive control.

[0045] Figure 4The 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 were studied; PBS was used as a negative control and 0.1% Triton X-100 was used as a positive control.

[0046] Figure 5 The study aimed to detect changes in fluorescence intensity of Staphylococcus aureus and Escherichia coli after PI absorption induced by cyclic peptide antibiotic 41 using flow cytometry; PBS was used as a negative control.

[0047] Figure 6 Fluorescent images of Staphylococcus aureus and Escherichia coli after laser confocal microscopy detection of cyclic peptide antibiotic 41 and incubation with a mixture of PI and Hoechst 33342 were obtained; PBS was used as a negative control.

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

[0049] Figure 8 is The number of corneal colonies and the results of H&E staining of corneal tissue sections after treatment with cyclic peptide antibiotics 7 and 41 for MRSA-infected keratitis in mice.

[0050] Figure 9 The results of H&E staining of lung tissue sections in mice treated with cyclic peptide antibiotics 7 and 41 for MRSA-infected pneumonia are shown. Vancomycin was used as a positive control.

[0051] Figure 10 Tests were conducted on the anti-fouling ability of Staphylococcus aureus after cyclic peptide antibiotic 11 was grafted onto the surface of contact lenses. Detailed Implementation

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

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

[0054] The instruments used in this invention include: a circular decolorizing shaker (SK-O330, Dalong Xingchuang Experimental Instruments (Beijing) Co., Ltd., China), a rotary evaporator (560-00000-00-0, Heidolph, Germany), a time-of-flight mass spectrometer (Maxis 4G, Bruker, Germany), a preparative HPLC (NU3001, Hanbang Technology, China), an analytical chromatograph (A02996, Waters, USA), and a freeze dryer (CoolSafe 55-4, Gene Company). Limited, China) Constant Temperature Shaker (IS-RDD3, Suzhou Jiemei Electronics Co., Ltd., China), Biosafety Cabinet (BSC-1500ⅡB2-X, Jinan Xinbeixi Biotechnology Co., Ltd., China), Constant Temperature Incubator (LT-BIX300M, Lidetech (Shanghai) Scientific Instruments Co., Ltd.), Benchtop Centrifuge (Thermo 75004250, Thermo Fisher Scientific, USA), Multifunctional Microplate Reader (FlexStation III, Meigu Molecular Instruments, USA), Carbon Dioxide Cell Culture Incubator (CLM-170B-8-TC, Taicang Yisigao Medical Devices Technology Co., Ltd., China), Fluorescence Microscope (U-LH50HG, Olympus Corporation, Japan), Micro-volume Nucleic Acid and Protein Analyzer (E113192, Implen GmbH (Germany), Mouse laryngoscope and nebulizer (Yuansengkaide Biotechnology Co., Ltd., China), Tissue cryo-morcerator (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), purchased from Heinz Technologies (China). N,N-dimethylformamide (DMF) was supplied by BASF (Germany). Dysmartin oxidant, N,N'-carbonyldiimidazole, and sodium borohydride were supplied by Merrill Technologies. Rink Amide MBHA resin was supplied by Lanxiao Technology (Xi'an, China). Acetonitrile was supplied by Shanghai CINC High Purity Solvents Co., Ltd. (Shanghai, China). D,L-type amino acids and cyclophosphamide were purchased from Merrill Chemical Technology Co., Ltd. Diethyl 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)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Tianjin Guangfu Reagent Co., Ltd. MH broth medium was purchased from Qingdao High-Tech Park Haibo Biotechnology Co., Ltd., and PBS buffer was purchased from Wuhan Saiweier Biotechnology Co., Ltd. Triton X-100 was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. RPMIDMEM medium was purchased from Shanghai Datashire Biotechnology Co., Ltd. Fetal bovine serum (FBS) was purchased from Shanghai Datashire 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 involves two steps: aldehyde-producing resin synthesis and peptide synthesis.

[0058] 1.1 Synthesis of aldehyde-producing resins (taking amic acid as an example):

[0059] First, Fmoc-valine was synthesized from Fmoc-valine. It was then reacted with serine to generate 2-(((9H-fluorene-9-yl)methoxy)carbonyl)amino)-2-methylpropyl)oxazolidine-4-carboxylic acid. This compound was then protected with BOC anhydride, and finally linked to MBHA resin via 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 involves first following the classic Fmoc solid-phase peptide synthesis method, where Fmoc-protected amino acids are sequentially linked to the synthesized aldehyde resin to form a linear peptide chain. Then, a cleavage reagent (prepared in the following ratio: TFA:DCM:TIS:H2O = (1-30):(70-99):(0-5):(0-5)) is used to remove the side chain protection, releasing the linear peptide aldehyde from the resin. The C-terminal aldehyde then undergoes an intramolecular cyclization reaction with the N-terminal cysteine ​​residue to form a thiazolidinyl ring, thus forming a cyclic peptide. The second strategy involves first following the classic Fmoc solid-phase peptide synthesis method, where Fmoc-protected amino acids are sequentially linked to the synthesized aldehyde resin to form a linear peptide chain. The peptides were sequentially linked to 2-chlorotriphenylmethyl chloride resin. Then, the peptides were cleaved from the resin using a cleavage agent (TFE:DCM = 30:70). The cleaved peptides were added to DMF, and condensing agents HOBT, HBTU, and DIEA were added to cyclize the peptides into amides. After cyclization, the peptides were washed with ethyl acetate and water, and the organic phase was collected. The organic phase was removed by rotary evaporation to obtain a white solid. The side chain protection was removed by adding a cleavage reagent (prepared in the following ratio: TFA:DCM:TIS:H2O = (1-30):(70-99):(0-5):(0-5)) to obtain a cyclic peptide antibiotic.

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

[0063] The synthesized cyclic peptide antibiotic was separated and purified by high-performance liquid chromatography (HPLC). Simply, the crude cyclic peptide sample was washed with diethyl ether to precipitate the precipitate, and the remaining diethyl ether was removed using a rotary evaporator. The precipitate was then 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 cyclic peptide antibiotic.

[0064] Example 3: In vitro antibacterial activity assay of cyclic peptide antibiotics

[0065] Cyclic peptide antibiotics 1-77 were synthesized according to the process described in Example 1 and purified according to the purification method described in Example 2. The strains used to determine the in vitro antibacterial activity of the cyclic peptide antibiotics and control antibiotics included standard strains (including Escherichia coli (ATCC 25922), C. albicans (ATCC 14053), and Staphylococcus 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 by the standard two-fold dilution method recommended by the National Committee for Clinical 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 subcultured 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, and 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 and cultured 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: Assay 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 pairs of mammalian erythrocytes (HC). 50 >100μg / mL)

[0073] The OD values ​​of erythrocyte solutions after treatment with cyclic peptide antibiotics 7, 9, 24, 25, 31, 37, 41, 43, 46, 48, 53, 55, 60, 61, 67, 70, and 76 were measured using an enzyme-linked immunosorbent assay (ELISA) reader. 490 The hemolytic activity of the cyclic peptide antibiotic was evaluated. The red blood cells used were obtained from mouse eyeballs after ischemic centrifugation.

[0074] First, whole blood from mice was centrifuged at 1200 rpm for 5 min, and the supernatant was discarded. After washing with PBS, 100% red blood cells were collected and diluted to 8% before being added to a 96-well plate. The plate was then incubated with the cyclic peptide antibiotic (100 μg / mL) in a cell culture incubator. After 1 hour, the 96-well plate was centrifuged at 1500 rpm for 15 min, and the supernatant was transferred to another 96-well plate. OD was measured using a microplate reader. 490 Finally, according to the formula: Hemolysis rate (%) = (Experimental group OD) / (Experimental group OD) 490 - Negative control group OD 490 ) / (Positive control group OD 490 - Negative control group OD 490 Calculate the hemolysis rate of each cyclic peptide antibiotic by multiplying the result by 100%.

[0075] like Figure 1 As shown, the hemolysis rate of cyclic peptide antibiotic 9 exceeded 20%, while other cyclic peptide antibiotics showed weak erythrocyte toxicity. The hemolysis rates of cyclic peptide antibiotics 24, 25, and 37 ranged from 10% to 20%, while the hemolytic toxicity of cyclic peptide antibiotics 7, 31, 41, 43, 46, 48, 53, 55, 60, 61, 67, 70, and 76 ranged from 1% to 10%.

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

[0077] Using cyclic peptide antibiotics 7, 41, 48, 53, 55, 60, 61, 67, 70, and 76 as representatives, the effect of cyclic peptide antibiotics on the growth and proliferation of Staphylococcus aureus ATCC 25923 at different time intervals was determined by colony-forming unit (CFU) counting method. The bactericidal kinetics of the cyclic peptide antibiotics were then represented by plotting the time interval on the x-axis and the logarithm of the colony count per milliliter on the y-axis. Simply put, the logarithmic phase concentration of 1.0 × 10⁻⁶ was used... 6CFU / mL of Staphylococcus aureus was added to a 96-well plate, followed by the addition of cyclic peptide antibiotics (final concentration 4×MIC). The plates were co-cultured at 37°C. At time intervals of 0h, 1 / 6h, 1 / 2h, 1h, 2h, 4h, 8h, and 12h, appropriate amounts of bacterial suspension were taken out, diluted appropriately, and 100μL of the diluted bacterial suspension was evenly spread onto MH solid medium using a spreader. After incubation overnight, colony counting was performed.

[0078] Figure 2 The results showed that at a concentration of 4×MIC, cyclic peptide antibiotics 7, 48, 55, 61, 67, and 76 could kill bacteria within 2 hours, while cyclic peptide antibiotics 41, 53, 60, and 70 could kill bacteria within 4 hours, all demonstrating rapid bactericidal ability.

[0079] Example 7. Determination of cyclic peptide antibiotic resistance

[0080] The potential of cyclic peptide antibiotics to induce bacterial resistance was assessed by determining the minimum inhibitory concentration (MIC) of Staphylococcus aureus ATCC 25923 in 21 consecutive determinations. Simply, Staphylococcus aureus in its logarithmic growth phase was diluted and added to a 96-well microtiter plate. The MIC was determined using the classic two-fold dilution method, and the concentration in the first well where no turbidity was observed was recorded as the initial MIC0. Then, 1 / 2 × MIC0 of the bacterial culture was used as the culture for the next MIC determination, and the first MIC was determined in the same manner and recorded as MIC1. Subsequently, the MIC values ​​of cyclic peptide antibiotics against Staphylococcus aureus were determined continuously in the same manner for 21 consecutive determinations, and the results were recorded as MIC1. 21 Finally, a graph was plotted with the number of measurements on the x-axis and the ratio of the MIC value to the MIC0 value for each measurement on the y-axis to represent the assessment of bacterial resistance induced by cyclic peptide antibiotics. The clinically used antibiotic Amoxicillin was used as a control.

[0081] like Figure 3 As shown, in the 21-day bacterial resistance test, the MIC values ​​of cyclic peptide antibiotic 7 were 3.125 μg / mL, cyclic peptide antibiotic 41 was 3.125 μg / mL, cyclic peptide antibiotic 48 was 6.25 μg / mL, cyclic peptide antibiotic 53 was 6.25 μg / mL, cyclic peptide antibiotic 55 was 3.125 μg / mL, cyclic peptide antibiotic 60 was 6.25 μg / mL, cyclic peptide antibiotic 61 was 6.25 μg / mL, cyclic peptide antibiotic 67 was 6.25 μg / mL, cyclic peptide antibiotic 70 was 6.25 μg / mL, and cyclic peptide antibiotic 76 was 12.5 μg / mL. These values ​​represented a 1-2 fold change from the initial MIC values, indicating that bacteria did not develop resistance.

[0082] The initial MIC for 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 effects of cyclic peptide antibiotics on bacterial cell membranes were assessed by detecting the disruptive effects of cyclic peptide antibiotics, represented by 7,41, on the cell membrane potential of Staphylococcus aureus ATCC 25923 and the induced PI uptake in Staphylococcus aureus.

[0085] 8.1 The disruptive effect of cyclic peptide antibiotics on the membrane potential of Staphylococcus aureus

[0086] The disruptive effect of cyclic peptide antibiotics on the membrane potential of Staphylococcus aureus was determined by the DiOC2(3) fluorescent probe method. Simply, Staphylococcus aureus ATCC 25923 was incubated at 37°C for 18 hours, 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, 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, a bacterial suspension containing DiOC2(3) (50 μL / well) was added to a 96-well microtiter plate, followed by 50 μL of cyclic peptide antibiotics of different concentrations (ranging from 1 to 8 times the MIC), and incubated for 30 minutes. Finally, the fluorescence of each well was detected using a microplate reader (Synergy NEO2, Agilent), 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 disrupt bacterial membrane potential in a concentration-dependent manner, and can completely disrupt bacterial membrane potential at concentrations of 4×MIC and 8×MIC.

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

[0089] The disruptive effect of cyclic peptide antibiotics on bacterial cell membrane integrity was assessed by measuring the uptake of the fluorescent dye propidium iodide (PI) by Staphylococcus aureus (ATCC 25923) after treatment with cyclic peptide antibiotics. Simply, after overnight incubation of Staphylococcus aureus at 37°C, the bacterial culture was centrifuged at 5000 rpm for 10 minutes, washed three times with PBS (10 mM, pH 7.4), and resuspended to 1 × 10⁻⁶. 9 The concentration of CFU / mL was determined. Subsequently, 100 μL of Staphylococcus aureus suspension was thoroughly mixed with an equal volume of cyclic peptide antibiotic solution and incubated at 37°C in a shaker for 1 hour. Then, 10 μL of PI (1 mg / mL) was added and incubated in the dark for 15 minutes for flow cytometry analysis (NovoCyte Quanteon, Agilent). The untreated group served as a negative control; alternatively, 10 μL each of PI (10 μM) and Hoechst 33342 (10 μM) were added and incubated in the dark for 15 minutes for fluorescence inverted microscopy imaging.

[0090] like Figure 5 As shown, the positive rate of PI (probiotic positivity) in Staphylococcus aureus treated with cyclic peptide antibiotic 41 at a concentration of 4×MIC was 98.27% by flow cytometry, 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 cyclic peptide 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 indicates that cyclic peptide antibiotic 41 can disrupt the bacterial cell membrane.

[0091] like Figure 6 As shown, Staphylococcus aureus treated with cyclic peptide antibiotic 41 exhibited significant red fluorescence, while no red fluorescence was detected in Staphylococcus aureus treated with PBS. Escherichia coli treated with cyclic peptide antibiotic 41 also exhibited significant red fluorescence, while no red fluorescence was detected in E. coli treated with PBS. This indicates that cyclic peptide antibiotic 41 can disrupt the bacterial cell membrane, leading to the entry of PI into the bacteria.

[0092] Example 9. Assay of the activity of cyclic peptide antibiotics in inhibiting Staphylococcus aureus biofilm formation.

[0093] The inhibition of Staphylococcus aureus (ATCC 25923) biofilm formation by cyclic peptide antibiotics was determined using the crystal violet method. Simply put, a concentration of 1.0 × 10⁻⁶ was used. 6Log-phase Staphylococcus aureus (CFU / mL) was added to a 96-well plate, followed by the addition of target cyclic peptide antibiotics 7 and 41 at concentrations ranging from 1×MIC to 16×MIC. The plates were then incubated at 37°C. After 24 hours, the surface-dwelling bacteria were removed from the 96-well plates, washed with PBS, fixed with methanol, stained with crystal violet solution for 15 min, and then dissolved in 95% ethanol to determine the OD (octane rating). 570 According to the formula, the biofilm formation inhibition rate (%) = [1 - (experimental group OD)] / [[1 - (experimental group OD)]]. 570 - Negative control group OD 570 ) / (Positive control group OD 570 - Negative control group OD 570 )]×100%.

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

[0095] Example 9. In vivo antibacterial activity assay of cyclic peptide antibiotics

[0096] The in vivo antibacterial activity of cyclic peptide antibiotics, represented by cyclic peptide antibiotics 7 and 41, was investigated. The in vivo antibacterial activity of cyclic peptide antibiotics was evaluated by establishing mouse models of MRSA-infected keratitis and mouse pneumonia, and their therapeutic effects in these animal models were examined.

[0097] 9.1 Mouse keratitis model

[0098] To test the in vivo antibacterial activity of cyclic peptide antibiotics in a mouse model of keratitis, Kunming mice were randomly divided into a negative control group, a cyclic peptide antibiotic 7 model group, a cyclic peptide antibiotic 41 model group, a cyclic peptide antibiotic 7 treatment group, a cyclic peptide antibiotic 41 treatment group, a linezolid control group, and a vancomycin control group. After anesthetizing the mice in the model group, treatment group, linezolid control group, and vancomycin control group, the cornea was scraped with a needle and then infected with 10 μL of MRSA bacterial suspension to establish a keratitis model. Twelve hours post-infection, mice in the cyclic peptide antibiotic 7 treatment group and the linezolid control group were treated with multiple eye drops of 10 μL of 1 mg / mL cyclic peptide antibiotic 7 and 1 mg / mL linezolid, respectively. Mice in the cyclic peptide antibiotic 41 treatment group and the vancomycin control group were treated with multiple eye drops of 10 μL of 1 mg / L cyclic peptide antibiotic 41 and 1 mg / mL vancomycin, respectively. Conversely, the model group received 10 μL of 0.9% saline each time. Eight hours after treatment, the mice were euthanized, and their eye tissues were collected for bacterial colony counting and H&E staining histological examination.

[0099] like Figure 8 is The results showed that the bacterial count in the mouse eyeballs treated with cyclic peptide antibiotic 7 (1 mg / ml) decreased by 0.854 log (p<0.05) compared to the model group, and was comparable to that in the linezolid (1 mg / ml) group (decreased by 0.864 log, p<0.05). H&E staining results indicated that the cornea in the model group had defects and a large number of inflammatory cells, while the corneal epithelial cells and basal layer in the cyclic peptide antibiotic 7 and linezolid treatment groups were relatively intact, with fewer inflammatory cells. The bacterial count in the mouse eyeballs treated with cyclic peptide antibiotic 41 (1 mg / ml) decreased by 1.244 log (p<0.05) compared to the model group, and was comparable to that in the vancomycin (1 mg / ml) group (decreased by 1.305 log, p<0.05). H&E staining results indicated that the corneal surface in the model group was rough, with a large number of inflammatory cells, while the corneal epithelial cells and basal layer in the cyclic peptide antibiotic 41 and vancomycin treatment groups were relatively smooth, with fewer inflammatory cells.

[0100] 9.2 Mouse pneumonia model

[0101] In determining the therapeutic potential of cyclic peptide antibiotics in a mouse model of pneumonia, female Kunming mice were randomly assigned to an untreated control group, a model group, a cyclic peptide antibiotic 7 treatment group, a cyclic peptide antibiotic 41 treatment group, and a vancomycin control group. After anesthetizing mice in the model group, cyclic peptide antibiotic treatment group 1, cyclic peptide antibiotic treatment group 2, and vancomycin control group, 25 μL of OD25 was administered via nebulizer needle under visual guidance. 600A 0.35 μL MRSA (ATCC 43300) bacterial suspension was injected into the trachea of ​​mice. Two and twelve hours post-infection, mice in the model group, the cyclic peptide antibiotic 7 treatment group, the cyclic peptide antibiotic 41 treatment group, and the vancomycin control group were treated with intraperitoneal injections of saline, cyclic peptide antibiotics 7 and 41 (5 mg / kg), and vancomycin (5 mg / kg), respectively. Twenty-four hours post-infection, all mice were sacrificed, and bacterial colony counting and H&E staining histological examination were performed.

[0102] like Figure 9 As shown, the cyclic peptide antibiotic 7 treatment group (5 mg / kg) effectively reduced the pulmonary bacterial load within 24 hours (reduction of 0.56 log, p<0.05), comparable to the vancomycin (5 mg / kg) treatment group (reduction of 0.76 log, p<0.05). H&E staining results showed that the alveoli in the cyclic peptide antibiotic 7 treatment group were intact, with no obvious inflammatory cells or capillary congestion observed, similar to the lung characteristics of the vancomycin group and the uninfected group. In contrast, the model group showed alveolar wall capillary congestion and an increase in inflammatory cells. The cyclic peptide antibiotic 41 treatment group (5 mg / kg) effectively reduced the pulmonary bacterial load within 24 hours (reduction of 0.62 log, p<0.05), comparable to the vancomycin (5 mg / kg) treatment group (reduction of 0.54 log, p<0.05). H&E staining results showed that the alveolar morphology of the cyclic peptide antibiotic 41 treatment group was intact, and no obvious inflammatory cells and capillary congestion were observed. The lung characteristics were similar to those of the vancomycin group and the uninfected group. In contrast, the alveolar walls of the model group were thickened, the alveolar tissue was reduced, the capillary congestion was observed, 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 representative implants to evaluate the antibacterial potential of cyclic peptide antibiotics grafted onto the implant surface. First, (3-aminopropyl)trimethoxysilane was attached to the hydroxyl groups on the contact lens surface. Then, 6-azidohexanoic acid was attached to (3-aminopropyl)trimethoxysilane via an amide condensation reaction. Subsequently, the alkynyl group containing cyclic peptide antibiotic 11 was attached to the azide group on the contact lens surface via a click chemistry reaction, completing the grafting of the cyclic peptide antibiotic onto the contact lens surface. Characterization was performed using X-ray photoelectron spectroscopy (XPS) (Kratos AXIS Ultra DLD, UK). Finally, the antibacterial effect was assessed by co-incubating the contact lenses grafted with cyclic peptide antibiotic 11 with a Staphylococcus aureus suspension and evaluating the bacterial colony count on the lens surface. Ungrafted contact lenses served as negative controls.

[0105] like Figure 10As shown, contact lenses grafted with cyclic peptide antibiotic 11 exhibited a 63% (p<0.05) reduction in bacterial adhesion compared to the ungrafted group, demonstrating the significant 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 and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. Cationic cyclic peptide antibiotic based on Lugdunin, characterized in that, The structural general formula of the cationic cyclic peptide antibiotic is shown as formula I: (Formula I); The amino acids in the cationic cyclic peptide antibiotic appear in L-, D- type or D-, L- type alternately. at least one of said R1, R3, R4groups is selected from any one of the following cationic groups: , , , , ; The other groups in the R1, R3, R4groups, excluding the selected cation group, are selected from any one of the following structures: , , , , , , , , , , , , , , , , , ; The R2group is selected from any one of the following structures: , , , , , , ; m=0-6, n=0-6, p=1-4, q=1 or 2, and m+n=3-6; o=1 or 2. The cationic cyclic peptide antibiotic is selected from the cyclic peptide antibiotics shown in the following formulae 1-77 or the salts of the cyclic peptide antibiotics shown in the following formulae 1-77: 。 2. Use of the cationic cyclic peptide antibiotic of claim 1 in the preparation of a medicament for the treatment of a drug-resistant bacterial infection.

3. Use according to claim 2, characterized in that, The drug-resistant bacteria are selected from at least one of Staphylococcus aureus, Escherichia coli, Candida albicans, Enterococcus faecalis, Enterococcus gallinarum, Bacillus subtilis or Staphylococcus epidermidis.

4. Use according to claim 2, characterized in that, The drug-resistant bacteria are selected from methicillin-resistant Staphylococcus aureus.

5. A medicament for treating a bacterial infection, characterized by, The active ingredient thereof comprises the cyclic peptide antibiotic of claim 1.

6. The medicament according to claim 5, characterized in that, It is used for treating, improving and / or preventing bacterial infections. It is used for treating, improving and / or preventing bacterial infections.

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