CN113082024A - Application of diguanylate cyclase inhibitor in medicine for inhibiting gram-negative bacteria and antibacterial medicine - Google Patents
Application of diguanylate cyclase inhibitor in medicine for inhibiting gram-negative bacteria and antibacterial medicine Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
Abstract
The invention relates to application of a diguanylate cyclase inhibitor in a medicine for inhibiting gram-negative bacteria and an antibacterial medicine, wherein the diguanylate cyclase inhibitor comprises one of ebselen or ebselen analogues, and the diguanylate cyclase inhibitor reduces the minimum inhibitory concentration of antibiotics to the bacteria under the concentration of not influencing the growth curve of the bacteria. Compared with the prior art, the invention greatly improves the capacity of the antibiotic for inhibiting gram-negative drug-resistant bacteria by utilizing the synergistic effect of the diguanylate cyclase inhibitor and the antibiotic, provides a novel sterilization and bacteriostasis method, has better bacteriostasis effect compared with the result of inhibiting the gram-negative drug-resistant bacteria by only depending on the antibiotic, can develop the analogue thereof for sterilization and bacteriostasis, and has the prospect of large-scale popularization.
Description
Technical Field
The invention relates to the field of antibacterial drugs, in particular to an application of a diguanylate cyclase inhibitor in a drug for inhibiting gram-negative bacteria and an antibacterial drug.
Background
Bacterial drug resistance seriously harms the public health in the world and causes huge economic loss. Overuse and abuse of antibiotics has led to the development of bacterial resistance. Some common gram-negative drug-resistant bacteria, such as escherichia coli producing extended-spectrum beta lactamase, multi-drug-resistant pseudomonas aeruginosa and the like, can become the most threatening drug-resistant bacteria without strict control and management. It is known that no new class of antibiotics has been approved for the inhibition of gram-negative bacteria for the last 50 years. The development of antibiotics has not kept pace with the evolution of bacteria and the development of resistance. Therefore, the new sterilization and bacteriostasis method is of great importance.
At present, more and more new bacteriostasis methods are based on the metabolism condition of bacteria, and the aim of sterilization is achieved by changing the metabolism level of the bacteria. For example, exogenous carbon source compounds are added to disturb central carbon metabolism, inhibit functional proteins related to lipopolysaccharide assembly of synthetic cell walls and the like, so that antibiotics can be assisted, and the sterilization effect is improved. Some sterilization methods based on inorganic materials, such as nano silver, have good sterilization effects and high durability, but the price and processing requirements of the materials themselves are relatively high. In addition, the nano silver can generate toxicity to nerves, skin and the like, and is not suitable for in vivo treatment. Natural antibacterial agents such as chitosan, while safe and harmless, are difficult to process and have a short service life.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide an application of a diguanylate cyclase inhibitor in a medicine for inhibiting gram-negative bacteria and a bacteriostatic medicine, and the small-molecule inhibitor is used for inhibiting enzyme activity related to the formation of bacterial drug resistance, so that the bactericidal ability of antibiotics is improved in an auxiliary manner.
The purpose of the invention can be realized by the following technical scheme:
the first purpose of the invention is to protect the application of a diguanylate cyclase inhibitor in the medicine for inhibiting gram-negative bacteria.
Further, the diguanylate cyclase inhibitor comprises one of ebselen or an ebselen analog. Ebselen may inhibit protein activity by forming covalent bonds between the thiols in the selenium and cysteine residues, and may also alter protein activity by its remaining chemical scaffold. Therefore, the ebselen analogs with proper scaffold structure and covalent modification with cysteine residue of diguanylate cyclase are all possible inhibitors.
Further, the diguanylate cyclase inhibitor reduces the minimum inhibitory concentration of the antibiotic to the bacteria at a concentration that does not affect the growth curve of the bacteria.
Further, the concentration of diguanylate cyclase inhibitor that does not affect the bacterial growth curve is obtained by:
s1: preparing stock solutions of antibiotics and diguanylate cyclase inhibitors;
s2: releasing the diguanylate cyclase inhibitor to a range of concentrations;
s3: culturing bacteria in a perforated plate, sequentially adding the diguanylate cyclase inhibitor obtained in the step S2 into the holes with bacteria liquid to serve as a series of experimental groups, and setting a control group to incubate the bacteria;
s4: and (3) performing data acquisition on all experimental groups and control groups in the S3 at intervals by using a microplate reader, and drawing bacterial growth curves under different concentrations of diguanylate cyclase inhibitors to obtain the concentration of the diguanylate cyclase inhibitors which have no influence on bacterial growth.
Further, the diguanylate cyclase inhibitor blocks the formation of a biofilm of gram-negative bacteria, so as to reduce the minimum inhibitory concentration of the antibiotic to the bacteria, wherein the minimum inhibitory concentration is obtained by the following steps:
s5: diluting the antibiotic solution obtained in S1 to a series of concentrations by a two-fold dilution method for antibiotic susceptibility testing;
s6: culturing bacteria in a perforated plate, sequentially adding the antibiotics obtained in S5 into the holes with bacteria liquid, sequentially adding the diguanylate cyclase inhibitor with the concentration obtained in S4 as a series of experimental groups, and simultaneously setting a control group for incubating the bacteria;
s7: and (4) determining the minimum inhibitory concentration of the experimental group and the control group in the S6 by using a microplate reader.
Further, the gram-negative bacteria are Escherichia coli capable of producing extended-spectrum beta lactamase.
Further, the gram-negative bacteria inhibiting drug is a beta lactam antibiotic.
Further, the antibiotic is ceftriaxone sodium, and the concentration of diguanylate cyclase inhibitor which does not affect the bacterial growth curve is as follows: less than or equal to 100 mu M.
Further, under the synergistic action of 100 mu M of ebselen, the MIC value of ceftriaxone sodium to E.coli producing extended-spectrum beta-lactamase is 0.32mg mL-1。
Further, the diluent used in S2 is dimethyl sulfoxide.
Further, the concentration of bacteria in S3 is 1-8 × 105CFU mL-1。
Further, the control group in S3, which does not contain diguanylate cyclase inhibitor, is dimethyl sulfoxide of the same volume as the diguanylate cyclase inhibitor of the experimental group.
Further, the absorbance OD in S4595Or OD600。
Further, the control group in S6, which does not contain diguanylate cyclase inhibitor, is dimethyl sulfoxide of the same volume as the diguanylate cyclase inhibitor of the experimental group.
The second purpose of the invention is to protect a bacteriostatic drug for gram-negative bacteria, which is characterized in that the bactericidal drug comprises the diguanylate cyclase inhibitor, antibiotics, pharmaceutically acceptable auxiliary materials and carriers.
Compared with the prior art, the invention has the following advantages:
1) the invention determines the concentration of ebselen which does not affect the growth of bacteria, and confirms that the bactericidal effect is from the synergistic effect of the diguanylate cyclase inhibitor and antibiotics, but not from the diguanylate cyclase inhibitor itself.
2) The invention utilizes the synergistic effect of the diguanylate cyclase inhibitor and the antibiotic, greatly improves the capability of the antibiotic in inhibiting gram-negative drug-resistant bacteria, and provides a novel sterilization and bacteriostasis method.
3) The diguanylate cyclase inhibitor ebselen used in the technical scheme is a novel anti-inflammatory drug applied to clinic, but the technical scheme finds a new application, can develop an analogue thereof to kill and inhibit bacteria, and has a prospect of large-scale popularization.
4) The bacterial drug susceptibility test used in the invention has mature operation steps and low cost, and the required consumables are easy to obtain.
Drawings
FIG. 1 is a schematic diagram of the experimental procedure for the synergistic sterilization using the diguanylate cyclase inhibitor and the antibiotic in example 1.
FIG. 2 is a graph showing the growth curves of E.coli producing extended-spectrum beta-lactamase in the presence of optimal concentrations of ebselen and in the absence of ebselen.
FIG. 3 is a graph showing that optimal concentration of ebselen reduces the MIC of ceftriaxone sodium against E.coli producing extended-spectrum beta-lactamase.
Detailed Description
The origin of the concept of the present invention is that bacterial-based diguanylate cyclases are able to promote bacterial biofilm formation. Biofilm is a major obstacle to sterilization. The activity of the diguanylate cyclase inhibitor, such as ebselen, is inhibited, so that the formation of a biological membrane can be inhibited, and the bactericidal effect of antibiotics is improved.
According to the technical scheme, the optimal inhibitor concentration which has no influence on the growth of bacteria is screened through a bacteria culture experiment in the presence of the diguanylate cyclase inhibitor. And (3) performing a bacterial sensitivity experiment by using a microdilution method, and comparing the Minimum Inhibitory Concentration (MIC) results of the antibiotics without the inhibitors and the diguanylate cyclase inhibitors with optimal concentrations under the conditions of the same culture medium, the same bacterial species and the same concentration, so as to reflect the synergistic antibiotic sterilization effect of the diguanylate cyclase inhibitors. Compared with the result of inhibiting gram-negative drug-resistant bacteria by only depending on antibiotics, the invention has better antibacterial effect.
In the technical scheme, the concentration of the diguanylate cyclase inhibitor which does not influence the growth curve of bacteria is obtained by the following steps:
s1: preparing stock solutions of antibiotics and diguanylate cyclase inhibitors;
s2: releasing the diguanylate cyclase inhibitor to a range of concentrations;
s3: culturing bacteria in a perforated plate, sequentially adding the diguanylate cyclase inhibitor obtained in the step S2 into the holes with bacteria liquid to serve as a series of experimental groups, and setting a control group to incubate the bacteria;
s4: and (3) performing data acquisition on all experimental groups and control groups in the S3 at intervals by using a microplate reader, and drawing bacterial growth curves under different concentrations of diguanylate cyclase inhibitors to obtain the concentration of the diguanylate cyclase inhibitors which have no influence on bacterial growth.
In the technical scheme, the diguanylate cyclase inhibitor blocks the formation of a biological membrane of gram-negative bacteria so as to reduce the minimum inhibitory concentration of the antibiotic to the bacteria, and the acquisition process of the minimum inhibitory concentration is as follows:
s5: diluting the antibiotic solution obtained in S1 to a series of concentrations by a two-fold dilution method for antibiotic susceptibility testing;
s6: culturing bacteria in a perforated plate, sequentially adding the antibiotics obtained in S5 into the holes with bacteria liquid, sequentially adding the diguanylate cyclase inhibitor with the concentration obtained in S4 as a series of experimental groups, and simultaneously setting a control group for incubating the bacteria;
s7: and (4) determining the minimum inhibitory concentration of the experimental group and the control group in the S6 by using a microplate reader.
The invention is described in detail below with reference to the figures and specific embodiments.
Example 1
The embodiment provides an experimental process for improving antibiotic inhibition of escherichia coli producing extended-spectrum beta lactamase by combining synergistic effect of ceftriaxone sodium and double-ornithine cyclase inhibitor ebselen, which comprises the following steps:
preparation of a culture medium: weighing a certain amount of culture medium dry powder according to the instruction of a purchased tryptone soybean broth culture medium, adding a certain volume of deionized water, and performing high-temperature sterilization treatment.
Preparing an ebselen selenium stock solution: 10mM stock was prepared in DMSO and diluted to 500. mu.M, 100. mu.M, 50. mu.M, 10. mu.M, etc.
Preparing bacterial suspension: absorbing culture solution of Escherichia coli producing extended-spectrum beta-lactamase, and making it pass through enzyme labeling instrument (OD)595) The concentration was determined and the overnight-cultured broth was diluted to 5X 10 with tryptone soy broth6～8×106CFU mL-1。
Growth curve determination loading: each 100. mu.L of tryptone soy broth was pipetted into a sample well of a 96-well plate, and then different concentrations of ebselen were added to the wells in series at 500. mu.M, 100. mu.M, 50. mu.M, 10. mu.M, 0. mu.M (only solvent dimethyl sulfoxide), and finally 10. mu.L of the diluted bacterial suspension was pipetted into each well.
And (3) measuring a growth curve: using a microplate reader (OD)595) Detecting the absorbance of the sample in the 96-well plate every 1 hour, measuring the influence of different concentrations of ebselen on the growth of bacteria, and determining that 100 μ M or less concentration of ebselen has no inhibitory influence on the growth of bacteria, the results are shown in FIG. 2.
Preparing a ceftriaxone sodium stock solution: 100mg mL of sterile water-1The stock solution was diluted to 10.24mg mL in sequence by a double dilution method-1，5.12mg mL-1，2.56mg mL-1，1.28mg mL-1，0.64mgmL-1And (4) carrying out concentration equalization.
Loading for MIC determination: 100 μ L of tryptone soy broth containing ceftriaxone sodium at various concentrations were pipetted into sample wells of a 96-well plate, 1.1 μ L of 10mM ebselen (final concentration 100 μ M) was pipetted, and finally 10 μ L of diluted bacterial suspension was pipetted into wells containing working solution and control wells.
Culturing: and culturing the well-added 96-well plate in an incubator at 37 ℃ for 18-20 hours.
And (3) detecting the minimum inhibitory concentration: using a microplate reader (OD)595) The minimum inhibitory concentrations of the experimental group and the control group were detected and compared, and the results are shown in fig. 3. As shown in the figure, under the synergistic action of 100 mu M of ebselen, the MIC value of ceftriaxone sodium to Escherichia coli producing extended-spectrum beta lactamase is changed from 2.56mg mL-1The reduction is 0.32mg mL-1The bacteriostatic effect is greatly improved.
The above examples are only exemplified by the synergistic inhibition of the effects of ebselen in combination with ceftriaxone sodium on the production of extended spectrum beta lactamase-producing escherichia coli, and the present invention is also applicable to the synergistic inhibition of other antibiotics and other gram-negative resistant bacteria. Almost all bacteria can produce biofilms under certain conditions, preventing antibiotics from entering the interior, and thus have sufficient time to evolve drug-resistant genes. Diguanylate cyclase is used for assisting in the generation of biological membranes and exists in various gram-negative bacteria such as pseudomonas aeruginosa and the like. By inhibiting the formation of biological membranes, antibiotics with various sterilization mechanisms including protein synthesis inhibition, cell wall synthesis inhibition, nucleic acid synthesis inhibition and the like can reach target sites without hindrance, and high-efficiency sterilization effect is achieved. Therefore, the invention has certain universality.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (10)
1. An application of biguanylate cyclase inhibitor in preparing the medicines for inhibiting gram-negative bacteria is disclosed.
2. The use of an inhibitor for inhibiting a gram-negative bacterium according to claim 1, wherein the inhibitor of diguanylate cyclase comprises one of ebselen or an analogue of ebselen.
3. Use of an inhibitor in a medicament for inhibiting gram-negative bacteria according to claim 1, wherein the diguanylate cyclase inhibitor reduces the minimum inhibitory concentration of an antibiotic to the bacteria at a concentration that does not affect the growth curve of the bacteria.
4. Use of an inhibitor in a medicament for inhibiting gram-negative bacteria according to claim 3, wherein the concentration of diguanylate cyclase inhibitor that does not affect the bacterial growth curve is obtained by:
s1: preparing stock solutions of antibiotics and diguanylate cyclase inhibitors;
s2: releasing the diguanylate cyclase inhibitor to a range of concentrations;
s3: culturing bacteria in a perforated plate, sequentially adding the diguanylate cyclase inhibitor obtained in the step S2 into the holes with bacteria liquid to serve as a series of experimental groups, and setting a control group to incubate the bacteria;
s4: and (3) performing data acquisition on all experimental groups and control groups in the S3 at intervals by using a microplate reader, and drawing bacterial growth curves under different concentrations of diguanylate cyclase inhibitors to obtain the concentration of the diguanylate cyclase inhibitors which have no influence on bacterial growth.
5. The use of an inhibitor for inhibiting gram-negative bacteria according to claim 4, wherein the diguanylate cyclase inhibitor inhibits biofilm formation by gram-negative bacteria, thereby reducing the minimum inhibitory concentration of the antibiotic for the bacteria, the minimum inhibitory concentration being obtained by:
s5: diluting the antibiotic solution obtained in S1 to a series of concentrations by a two-fold dilution method for antibiotic susceptibility testing;
s6: culturing bacteria in a perforated plate, sequentially adding the antibiotics obtained in S5 into the holes with bacteria liquid, sequentially adding the diguanylate cyclase inhibitor with the concentration obtained in S4 as a series of experimental groups, and simultaneously setting a control group for incubating the bacteria;
s7: and (4) determining the minimum inhibitory concentration of the experimental group and the control group in the S6 by using a microplate reader.
6. The use of an inhibitor according to claim 1 in a medicament for inhibiting gram-negative bacteria, wherein the gram-negative bacteria are E.coli producing extended-spectrum beta lactamases.
7. The use of an inhibitor in a gram negative bacteria inhibiting medicament according to claim 2 wherein the gram negative bacteria inhibiting medicament is a beta lactam antibiotic.
8. Use of an inhibitor in a medicament for inhibiting gram-negative bacteria according to claim 7, wherein the antibiotic is ceftriaxone sodium and the concentration of diguanylate cyclase inhibitor that does not affect the bacterial growth curve is: less than or equal to 100 mu M.
9. The use of an inhibitor in a medicament for inhibiting gram-negative bacteria according to claim 8, wherein the MIC of ceftriaxone sodium to E.coli producing extended-spectrum beta-lactamase is 0.32mg mL under the synergistic effect of 100 μ M ebselen-1。
10. A bacteriostatic drug against gram-negative bacteria, characterized in that the bacteriostatic drug component comprises the diguanylate cyclase inhibitor of any one of claims 1 to 9, an antibiotic, and pharmaceutically acceptable excipients and carriers.
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| CHENG CHEN 等: "Ebselen bearing polar functionality: Identification of potent antibacterial agents against multidrug-resistant Gram-negative bacteria", 《BIOORGANIC CHEMISTRY》 * |
| 张月娟: "抗生素耐药靶蛋白金属 β-内酰胺酶及其耐药细菌的表征与抑制研究", 《中国优秀博硕士学位论文全文数据库(博士)医药卫生科技辑》 * |
| 陈成: "金属β-内酰胺酶及耐药细菌的抑制研究", 《中国优秀博硕士学位论文全文数据库(硕士)工程科技Ⅰ辑》 * |
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