CN111362834A - Antibacterial amidine oligomer with drug resistance and preparation method and application thereof - Google Patents
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Abstract
The invention discloses an antibacterial amidine oligomer with drug resistance and a preparation method and application thereof, wherein the antibacterial species of the amidine oligomer comprise bacillus subtilis, escherichia coli, enterococcus faecalis, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa and enterobacter cloacae. The structure disclosed by the invention has a dual antibacterial mechanism of destroying cell membranes and combining with chromosome DNA, has the characteristics of quick sterilization and drug resistance, and shows broad antibacterial spectrum. In addition, the structure utilizes the difference that the bacteria have no cell nucleus and the bacteria have the cell nucleus, specifically targets the DNA of the bacteria, obviously reduces the cytotoxicity and improves the therapeutic index of the bacteria.
Description
Technical Field
The invention relates to the field of pharmaceutical chemicals, in particular to an antibacterial amidine oligomer with drug resistance and a preparation method and application thereof.
Background
The problem of resistance to antibiotics has gradually become a new threat to human health care. Recently we have established the concept of anti-drug resistant antibacterials, aiming to cope with this huge threat by reducing the rate of development of drug resistance.
Antimicrobial agents with resistance to drugs should have the following characteristics: 1) the rate at which bacteria develop resistance to the drug is low during drug treatment; 2) has good effect of killing pathogens with multidrug resistance. It is well known that the root cause of antibiotic resistance is mutation of its target, rendering drugs of various mechanisms of action ineffective. Thus, if a drug can be targeted to multiple targets, or its target involves a complex biological process, the probability of developing drug resistance will be greatly reduced. Compounds of this type of antimicrobial polymers meet the concept of resistance to drugs. The main action mechanism of the antibacterial polymer is to destroy cell membranes, and the biosynthesis of the cell membranes relates to a complex biological process, so that the antibacterial polymer has the characteristics of low drug resistance generation rate and resistance to multi-drug resistant bacteria, and meets the requirements of a drug resistant antibacterial agent. However, the similarity of the structures of the bacterial cell membrane and the mammalian cell membrane makes the antibacterial polymer have high cytotoxicity and hemolytic activity, resulting in large side effects. Thus, to date, there is no FDA approved antimicrobial polymer for use in the treatment of bacterial infections.
Disclosure of Invention
In order to solve the problems, the invention discloses an antibacterial amidine oligomer with drug resistance, a preparation method and application thereof. The structure disclosed by the invention has a dual antibacterial mechanism of destroying cell membranes and combining with chromosome DNA, has the characteristics of quick sterilization and drug resistance, and shows broad antibacterial spectrum. In addition, the structure utilizes the difference that the bacteria have no cell nucleus and the bacteria have the cell nucleus, specifically targets the DNA of the bacteria, obviously reduces the cytotoxicity and improves the therapeutic index of the bacteria.
In order to achieve the technical effects, the technical scheme of the invention is as follows:
an antimicrobial amidine oligomer having resistance to drugs, said amidine oligomer having the formula:
wherein n is more than or equal to 5 and less than or equal to 8;
wherein the molecular formula of R comprises one or more of formula (I), formula (II), formula (III), formula (IV), formula (V), formula (VI), formula (VII) and formula (VIII);
in a further improvement, R in the molecular formula of the amidine oligomer is shown as a formula (IV).
A preparation method of an antibacterial amidine oligomer with drug resistance comprises the following steps: weighing terephthalic acid diimine hydrochloride and H2N-R-NH2Uniformly mixing with anhydrous N, N-dimethylformamide DMF, adding triethylamine TEA, stirring for 84-96h at 30-40 ℃ under the protection of inert gas to obtain a mixture, adjusting the pH of the mixture to 1-2, then intercepting to obtain a substance with the molecular weight of more than 1.5KDa, and freeze-drying to obtain the amidine oligomer;
wherein R is shown as formula (I), formula (II), formula (III), formula (IV), formula (V), formula (VI), formula (VII) or formula (VIII);
the further improvement comprises the following steps:
weighing terephthalic acid diimmonium ester hydrochloride and 1, 4-bis (3-aminopropyl) piperazine, then adding and uniformly mixing with anhydrous N, N-dimethylformamide DMF, adding triethylamine TEA, stirring for 84-96h at 30-40 ℃ under the protection of inert gas to obtain a mixture, adjusting the pH of the mixture to 1-2, then intercepting to obtain a substance with molecular weight more than 1.5KDa, and freeze-drying to obtain amidine oligomer; wherein the molar ratio of the phthalic acid diimmonium ester hydrochloride, the 1, 4-bis (3-aminopropyl) piperazine and the TEA is 1: 1: 4.
in a further improvement, the terephthalic acid diimine hydrochloride comprises one or any mixture of iron terephthalate diimine hydrochloride, aluminum terephthalate diimine hydrochloride and lithium terephthalate diimine hydrochloride.
Use of the above antibacterial amidine oligomer having resistance to drugs for use as an antibacterial agent.
In a further improvement, the amidine oligomer is used for antibiosis, and the antibacterial species of the amidine oligomer comprise bacillus subtilis, escherichia coli, enterococcus faecalis, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa and enterobacter cloacae.
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FIG. 1 is a schematic diagram of the reaction of the present invention;
FIG. 2 is a diagram of a flow experiment demonstrating that PBAM-4 has a strong membrane rupture capability;
fig. 3 is an SEM image: shows that the PBAM-4 treated cell membrane surfaces of escherichia coli and acinetobacter baumannii both have shrinkage of different degrees;
FIG. 4 shows a graph of PBAM-4 with binding to bacterial genomic DNA showing a significant increase in particle size after incubation of 50. mu.g/mL PBAM-4 with different concentrations of E.coli genomic DNA (0.05pmol/mL and 0.25 pmol/mL);
FIG. 5 is a graph showing the effect of added DNA on the antibacterial activity of a compound.
FIG. 6 is a profile of FITC-labeled PBAM-4 in B.subtilis (top) and E.coli (bottom); PBAM-4 is mainly distributed in the cell membrane and nucleus of bacterial cells;
FIG. 7 is a profile of rhodamine-labeled PBAM-4 in NIH/3T3 cells; PBAM-4 is distributed primarily within the cytoplasm of mammalian cells;
FIG. 8 is a graph comparing the rates of drug resistance development after 576 generations of E.coli were treated with kanamycin and PBAM-4, respectively;
FIG. 9a is a photomicrograph of normal NIH/3T3 cells;
FIG. 9b 1.24 × 109CFU/mL of bacteria infected NIH/3T3 cell micrographs, cells were killed by rapidly proliferating bacteria;
FIG. 9c a micrograph of 10. mu.g/mL PBAM-4 treated bacterially infected NIH/3T3 cells, demonstrating that PBAM-4 can effectively kill bacteria in a co-culture model, achieving the effect of rescuing cells;
FIG. 9d microscopic picture of 10. mu.g/mL PBAM-4 treated bacteria infected NIH/3T3 cells, demonstrating that it does not affect the morphology of the cells and has good biocompatibility;
FIG. 10 model of bacteria co-culture with erythrocytes. PBAM-4 was able to rescue efficiently erythrocytes co-cultured with bacteria, without producing a significant hemolytic effect, compared to small molecule antibiotics;
FIG. 11 is a graph of in vivo antibacterial activity of PBAM-4 evaluated using a model of bacterial nematode infection, in which PBAM-4 can efficiently and completely kill the three multidrug resistant strains (Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus) infected by nematodes, compared to antibiotics (gentamicin, ciprofloxacin, and ampicillin) having a better effect on these bacteria in vitro;
FIG. 12a is a graph of the antibacterial activity study of PBAM-4 in a mouse epidermal infection model;
FIG. 12b is a graph of the increased survival of infected mice by PBAM-4, data analyzed by student's t-test, statistically significant differences indicated by asterisks,. P < 0.05;
FIG. 12c is a bacterial map showing that PBAM-4 can completely kill the mouse infected site with high efficiency as compared with the control group PBS and ciprofloxacin.
Detailed Description
The technical solution of the present invention is described in detail below by means of specific embodiments and with reference to the attached drawings, and the components or devices in the following embodiments are all general standard components or components known to those skilled in the art, and the structure and principle thereof can be known to those skilled in the art through technical manuals or through routine experiments.
Example 1
Through tests, the antibacterial amidine oligomer with broad-spectrum antibacterial property in 8 is developed, and the chemical formula is shown as follows:
wherein n is more than or equal to 5 and less than or equal to 8;
wherein the molecular formula of R is shown as formula (I), formula (II), formula (III), formula (IV), formula (V), formula (VI), formula (VII) or formula (VIII);
for convenient recording, the compounds of formula (I), formula (II), formula (III), formula (IV), formula (V), formula (VI), formula (VII) and formula (VIII) of R are respectively numbered as PBAM-1, PBAM-2, PBAM-3, PBAM-4, PBAM-5, PBAM-6, PBAM-7 and PBAM-8. The antibacterial properties of the 8 compounds are shown in tables 1 and 2:
antibacterial Properties of the compounds in Table 18
Antibacterial property of Table 28 Compounds
Wherein the content of the first and second substances,aindicates not tested;bhalf the hemolysis rate;c,d,e,f,g,hthe different mechanisms of action of antibiotics are:cinhibiting synthesis of bacterial cell walls;d,etargeting 30s and 50s ribosomes, respectively, to inhibit protein synthesis;faffecting nucleic acid synthesis and replication;gaffect the metabolism of folic acid;hdestroying the cell membrane.
The following will explain PBAM-4 having the best antibacterial property:
materials and methods: all reagents were supplied by Acros, TCI (USA), Sigma-Aldrich, Michelin, Adamax et al organic reagents and Byunnan Biotech, all used without further purification (among others). Ultrapure water for the experiments was obtained from a Milli-Q purification instrument. The inert gas is nitrogen or argon.
The synthetic synthesis procedure for PBAM-4 is shown in FIG. 1. Terephthalic acid diimmonium ester hydrochloride (43.9mg,0.15mmol,1eq.) and 1, 4-bis (3-aminopropyl) piperazine (30.0mg, 0.15mmol,1eq.) were weighed into a 7mL glass bottle, 1.5mL of anhydrous DMF was added and mixed well, and 83.7. mu.L of TEA (0.6mmol,4eq.) was added. Stirring for 96h at 35 ℃ under the protection of inert gas. Adding dilute hydrochloric acid solution to the mixture to adjust pH to 1-2, dialyzing in dialysis bag with molecular weight cutoff of 1.5kDa for 10 hr, and changing water every two hours. After lyophilization, 36mg of a white solid was obtained.
PBAM-4 has a broad spectrum of antibacterial activity we tested PBAM-4 for antibacterial activity against a variety of bacteria (tables 1 and 2). PBAM-4 showed good antibacterial activity with all tested minimum inhibitory concentrations in the lower ug/mL range. Besides, PBAM-4 was tested for activity against 12 highly resistant clinical pathogens, and the minimal inhibitory concentrations were found to be in the lower ug/mL range. These broad spectrum antibacterial activities clearly indicate that PBAM-4 has resistance properties. In addition, PBAM-4 showed a lower hemolytic activity towards erythrocytes with a half-maximal hemolytic concentration of more than 5000. mu.g/mL. Using the accepted algorithm for the therapeutic index of antimicrobial polymers (therapeutic index ═ half maximal hemolytic concentration/minimum inhibitory concentration), PBAM-4 has a therapeutic index greater than 2500 for most bacteria. The minimum bactericidal concentration values of all bacteria are extremely consistent with the corresponding minimum bacteriostatic concentration, which indicates that PBAM-4 have strong bactericidal action.
Validation of membrane targeting mechanism we first evaluated the membrane rupture ability of PBAM-4 using flow experiments. In flow experiments, Propidium Iodide (PI) is used as a fluorescent dye to assess the integrity of bacterial cell membranes. After centrifugation of E.coli cultured to stationary phase, the medium was discarded, washed three times with PBS, and then resuspended to OD with 100. mu.M PI-containing PBS600nmThe cells were incubated at 37 ℃ for 4h with the addition of PBAM-4 and ciprofloxacin at the indicated concentrations, 0.1. The samples were analyzed using a BD Accuri C6 Plus flow cytometer. Since Propidium Iodide (PI) can only enter cells with damaged cell membranes, PI can be a good indicator of the integrity of bacterial cell membranes in flow experiments. As shown in FIG. 2, the results indicate that E.coli treated with PBAM-4 accumulated more PI and thus emitted more fluorescence. Untreated E.coli and E.coli treated with the non-membrane-targeting antibiotic ciprofloxacin showed very low fluorescence.
We further used scanning electron microscopy to demonstrate the membrane rupture capability of PBAM-4. As shown in FIG. 3, bacteria treated with PBAM-4 showed significant membrane disruption and shrinkage relative to untreated E.coli and A.baumannii controls.
Validation of binding to DNA antibacterial mechanism the oligomer was designed based primarily on a dual antibacterial mechanism of membrane targeting and DNA targeting. In addition to the demonstration of the membrane-breaking ability of PBAM-4, we also demonstrated that this oligomer has the ability to bind bacterial DNA. Dynamic Light Scattering (DLS) studies showed that different concentrations of PBAM-4 can promote aggregation of E.coli genomic DNA, as shown in FIG. 4. Binding DNA to form an oligomer-DNA complex provides strong evidence for an affinity between PBAM-4 and DNA. In addition, the antibacterial activity of PBAM-4 is inhibited under the conditions of DNA addition. However, the small molecule antibiotic polymyxin targeted to the cell membrane did not have a reduction in antibacterial activity under this condition, as shown in fig. 5. This phenomenon can be explained by the partial inactivation of the antibacterial activity of PBAM-4 after it binds to DNA.
After determining that PBAM-4 has the ability to bind to DNA in vitro, we observed the binding of bacterial DNA to cellular DNA using fluorescein-labeled PBAM-4. FIG. 6 shows the staining of PBAM-4 on two bacteria, PBAM-4 staining channel ("PBAM-4"), cell membrane staining channel ("FM 4-64") and DNA staining channel ("DAPI") are well integrated, and PBAM-4 targets both cell membrane and non-cytoprotective DNA in bacteria. Unlike staining in bacteria, PBAM-4 (fig. 7, "PBAM-4" channel) in mammalian cells stays in the matrix and does not enter the nucleus (fig. 7, "DAPI" channel), probably because its large size is excluded from the nucleus by the nuclear membrane and thus cannot bind to the DNA of the cell, which is crucial to reduce its cytotoxicity. PBAM-4 exhibits high selectivity and therapeutic index because it binds to bacterial DNA but not mammalian DNA and kills bacteria much more than cells.
Determination of the rate of resistance development of PBAM-4 introduces an additional benefit of a dual antibacterial mechanism that can reduce the rate at which bacteria develop resistance to compounds. The membrane-targeting polymers, which target the cell membrane, are very complex in their biosynthesis and only when multiple random mutations occur simultaneously and result in changes in the membrane structure, reduce the sensitivity of bacteria to them, and are therefore considered as a class of antimicrobial compounds with low drug resistance. PBAM-4, one of the membrane-targeting polymers, also has low drug resistance characteristics. In addition, however, PBAM-4 has DNA as a secondary target, and thus the bacteria need to simultaneously mutate the membrane structure and the DNA structure to develop resistance to PBAM-4. Therefore, the dual mechanism of membrane targeting and DNA targeting endows PBAM-4 with the possibility of drug resistance, and can reduce the drug resistance generation rate to a lower level; and our experimental observations validate this hypothesis. As shown in FIG. 8, when Escherichia coli was treated repeatedly with kanamycin, the second minimum inhibitory concentration of the standard antibiotic, the strain developed a drug-resistant mutation, which showed that the minimum inhibitory concentration of kanamycin was increased continuously, and the minimum inhibitory concentration was increased 14-fold after the bacteria had replicated 576 generations. In contrast, PBAM-4 did not develop significant resistance under the same conditions. All the above results show that PBAM-4 has broad-spectrum antibacterial activity, and clearly reveal the nature of this novel antibacterial oligomer material against the generation of drug resistance.
Evaluation of antimicrobial efficacy using cell infection model we continued to evaluate the antimicrobial efficacy of PBAM-4 in the presence of mammalian cells. We first evaluated using NIH/3T3 cells as a model. In the case of co-culture of Acinetobacter baumannii and NIH/3T3, the mammalian cells that were originally morphologically normal (FIG. 9a) would be killed by the rapidly proliferating bacteria (FIG. 9 b). Infected NIH/3T3 cells were restored to the preinfection state by treatment with PBAM-4 (FIG. 9 c). As a control experiment, PBAM-4 was not significantly cytotoxic to NIH/3T3 cells (FIG. 9 d).
Similarly, excess bacteria can cause hemolytic effect of erythrocytes, while PBAM-4 can inhibit hemolysis of erythrocytes through its powerful bactericidal effect, as shown in FIG. 10, the number of bacteria was from 1.5 × 10 after the addition of 8. mu.g/mL PBAM-48CFU/mL was reduced to 0CFU/mL, while the erythrocyte hemolysis rate was reduced from 88% to 0%. The standard antibiotic meropenem used as a control had only a 10-fold reduction2CFU/mL of bacteria and a hemolysis rate of 23%. The experimental results show that PBAM-4 not only has good antibacterial activity, but also has good biocompatibility.
Antibacterial efficiency assessment by using a nematode infection model based on the excellent performance expressed by PBAM-4 in the in vitro studies, we continued to use a simple nematode model for preliminary assessment of its activity in vivo. In the study, we first infected nematodes with different strains, staphylococcus aureus, acinetobacter baumannii or pseudomonas aeruginosa. These infected nematodes were divided into three groups, PBS-treated group, standard antibiotic group, PBAM-4 treated group. After drug treatment, we ground the nematodes and tested the amount of bacteria in the broke. FIG. 11 clearly shows that PBAM-4 can efficiently kill bacteria and multidrug-resistant strains in a nematode infection model. In comparison, traditional antibiotics with moderate or better bactericidal activity in vitro have shown poor efficacy against multidrug resistant strains in this nematode model. The results of this simple in vivo model demonstrate the high efficacy of PBAM-4 in the treatment of bacterial infections, especially multidrug-resistant bacteria.
Evaluation of antibacterial Activity Using mouse wound model to better evaluate the activity of PBAM-4 in animal models, we evaluated the efficacy of PBAM-4 using a mouse precision wound model in which we made a 2cm × 2cm wound on the mouse epidermis and infected the wound surface with Pseudomonas aeruginosa, then the wound surface was treated with PBAM-4, ciprofloxacin and PBS, respectively, and observed wound recovery for 15 days (FIG. 12 a). it is clear that PBAM-4 significantly improved the survival rate of infected mice, reducing the mortality rate from 50% to 10% (FIG. 12 b). after PBAM-4 treatment, we examined the bacterial count of the wound and observed that PBAM-4 cleared all the bacteria from the wound (FIG. 12c), consistent with the rapid bactericidal kinetics of PBAM-4 and the lower MIC/MBC values.
In conclusion, the oligomer can simultaneously target the cell membrane and the nucleic acid of bacteria, generate a synergistic effect and have a higher therapeutic index. Thanks to its dual antimicrobial mechanism, the oligomer retains all the antimicrobial advantages of traditional antimicrobial polymers, such as fast bactericidal kinetics and lower probability of drug resistance; in addition, the selectivity provided by the DNA as a second target greatly improves the cytotoxicity, and provides a scheme for treating multidrug-resistant pathogens.
Example 2
To expand the variety of antibacterial amidine oligomers, different H's are used2N-R-NH2Preparation of antibacterial amidine oligomer as mixed raw materialThe obtained mixed product also has obvious antibacterial property, as shown in table 3:
table 3 antibacterial property of mixed raw materials for preparing antibacterial amidine oligomer
Table 3 shows that the antibacterial amidine oligomer has the same antibacterial performance when the R group is mixed.
The above description is only one specific guiding embodiment of the present invention, but the design concept of the present invention is not limited thereto, and any insubstantial modification of the present invention using this concept shall fall within the scope of the invention.
Claims (7)
1. An antibacterial amidine oligomer having resistance to drugs, wherein the amidine oligomer has the following formula:
wherein n is more than or equal to 5 and less than or equal to 8;
wherein the molecular formula of R comprises one or more of formula (I), formula (II), formula (III), formula (IV), formula (V), formula (VI), formula (VII) and formula (VIII);
2. the antimicrobial resistant amidine oligomer of claim 1 wherein R is of formula (IV).
3. A preparation method of an antibacterial amidine oligomer with drug resistance is characterized by comprising the following steps: weighing terephthalic acid diimine hydrochloride and H2N-R-NH2Uniformly mixing with anhydrous N, N-dimethylformamide DMF, adding triethylamine TEA, stirring for 84-96h at 30-40 ℃ under the protection of inert gas to obtain a mixture, adjusting the pH of the mixture to 1-2, then intercepting to obtain a substance with the molecular weight of more than 1.5KDa, and freeze-drying to obtain the amidine oligomer;
wherein R is shown as formula (I), formula (II), formula (III), formula (IV), formula (V), formula (VI), formula (VII) or formula (VIII);
4. the method for preparing the antibacterial amidine oligomer having resistance to drugs according to claim 3, comprising the steps of:
weighing terephthalic acid diimmonium ester hydrochloride and 1, 4-bis (3-aminopropyl) piperazine, then adding and uniformly mixing with anhydrous N, N-dimethylformamide DMF, adding triethylamine TEA, stirring for 84-96h at 30-40 ℃ under the protection of inert gas to obtain a mixture, adjusting the pH of the mixture to 1-2, then intercepting to obtain a substance with molecular weight more than 1.5KDa, and freeze-drying to obtain amidine oligomer; wherein the molar ratio of the phthalic acid diimmonium ester hydrochloride, the 1, 4-bis (3-aminopropyl) piperazine and the TEA is 1: 1: 4.
5. the method of producing an antibacterial amidine oligomer having resistance to drugs according to claim 4, wherein said diimine terephthalate hydrochloride comprises one or any mixture of iron, aluminum and lithium diimine terephthalate hydrochloride.
6. Use of an anti-drug-resistant antibacterial amidine oligomer according to any one of claims 1 to 5 as an antibacterial agent.
7. The use of an anti-drug resistant antibacterial amidine oligomer according to claim 6 for antibacterial use, wherein the antibacterial species of amidine oligomer include Bacillus subtilis, Escherichia coli, enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae.
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Cited By (3)
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| CN113754564A (en) * | 2021-08-02 | 2021-12-07 | 湖南大学 | Antibacterial amidine oligomer with anti-drug resistance and preparation method and application thereof |
| CN114773250A (en) * | 2021-08-02 | 2022-07-22 | 湖南大学 | Antibacterial amidine oligomer with anti-drug resistance and preparation method and application thereof |
| CN114933550A (en) * | 2021-08-02 | 2022-08-23 | 湖南大学 | Antibacterial peptide mimics with polyphenyl indole amidine and preparation method and application thereof |
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| CN111362834B (en) | 2021-04-02 |
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