CN110698457A - Inhibitor Lo-tert-butyl ester for resisting staphylococcus aureus virulence and biofilm formation and application thereof - Google Patents
Inhibitor Lo-tert-butyl ester for resisting staphylococcus aureus virulence and biofilm formation and application thereof Download PDFInfo
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Abstract
The invention provides an inhibitor Lo-tert-butyl ester for resisting staphylococcus aureus virulence and biofilm formation and application thereof, wherein the chemical structural formula of the inhibitor Lo-tert-butyl ester for resisting staphylococcus aureus virulence and biofilm formation is shown as a formula (1). The inhibitor Lo-tert-butyl ester for resisting staphylococcus aureus virulence and biofilm formation has an inhibiting effect on staphylococcus aureus virulence and biofilm formation, and can be used for preparing a medicament for inhibiting staphylococcus aureus virulence and biofilm formation, or preparing a medicament for treating diseases caused by staphylococcus aureus, or preparing a medical instrument or medical appliance disinfectant.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an inhibitor Lo-tert-butyl ester for resisting staphylococcus aureus virulence and biofilm formation and application thereof.
Background
Staphylococcus aureus can infect different parts of the human body to cause various infectious diseases, from common skin diseases such as folliculitis, acne and hordeolum to deep and fatal diseases such as pneumonia, endocarditis, osteomyelitis and other metastatic complications. In the process that the staphylococcus aureus invades a host and induces the host to be infected, various virulence factors such as hemolysin, extracellular protease, leukocidin, phenol soluble protein and the like can be secreted, so that the invasion and the damage of the staphylococcus aureus to the host are assisted, and further, the occurrence of diseases is induced. At present, with the wide use of antibacterial drugs, the emergence of drug-resistant bacteria, especially methicillin-resistant staphylococcus aureus (MRSA), brings difficulties to clinical treatment. Vancomycin and linezolid are few antibacterial drugs capable of treating MRSA infection at present, but at home and abroad, more and more vancomycin-insensitive staphylococcus aureus (VISA/hVISA) and linezolid drug-resistant strains are found, so that the selection of clinical antibacterial drugs is severely limited. In addition, staphylococcus aureus can be adhered to the surface of human tissue cells or medical implant materials to form a biofilm structure consisting of extracellular polysaccharide adhesion molecules, proteins, teichoic acid, extracellular DNA (eDNA) and the like, so that the sensitivity of bacteria to antibacterial drugs is reduced, attack and phagocytosis of host immune cells are avoided, and chronic infection and prolonged disunion are caused. The serious problem is that the infection in hospital related to staphylococcus aureus biofilm is increasing with the wide application of medical implant materials such as various catheters, dialysis technology, prosthetic joints and the like in recent years. At present, researches show that staphylococcus aureus resistant strains such as MRSA and the like also have stronger toxicity, can cause severe infectious diseases such as septic shock and endocarditis of patients to cause higher fatality rate, and also show that the resistant strains also have stronger biofilm formation capability. Therefore, in order to reduce the death of patients caused by the infection of the strains, inhibiting the virulence and biofilm formation of staphylococcus aureus becomes one of the difficulties and hot spots associated with bacteria to be urgently solved in recent years.
Disclosure of Invention
Aiming at the technical problems, the invention discloses an inhibitor Lo-tert-butyl ester for resisting staphylococcus aureus virulence and biofilm formation and application thereof, which have the functions of inhibiting staphylococcus aureus virulence and biofilm formation and have no obvious toxicity to mammalian cells.
In contrast, the technical scheme adopted by the invention is as follows:
an inhibitor Lo-tert-butyl ester for resisting staphylococcus aureus virulence and biofilm formation, which has the following chemical structural formula:
wherein Lo-tert-butyl ester has a molecular formula of C27H31CIN2O4The product is colorless and odorless transparent liquid at normal temperature, has the characteristics of high boiling point, good thermal stability and non-proton, and can be dissolved in most organic substances such as ethanol, propanol, benzene, chloroform and the like. The Lo-tert-butyl ester is a small molecular compound. The micromolecular compound Lo-tert-butyl ester can obviously inhibit the toxicity and biofilm formation of staphylococcus aureus in vitro and in vivo; the small molecular compound Lo-tert-butyl ester does not influence the growth of bacteria; has no obvious toxicity to mammalian cells and no obvious hemolytic effect to human erythrocytes.
In the invention, the small molecular compound Lo-tert-butyl ester can be used for preparing a medical instrument or medical appliance disinfectant, and can be further used for modifying a chemical structure to prepare a new medicament for resisting gram-positive bacteria infection.
The invention also discloses a medicine for resisting the toxicity of staphylococcus aureus and the formation of a biofilm, which comprises the inhibitor Lo-tert-butyl ester for resisting the toxicity of staphylococcus aureus and the formation of a biofilm.
Further, the medicine is liquid, and the concentration of the Lo-tert-butyl ester in the medicine is not less than 25 mu M.
The invention also discloses application of the Lo-tert-butyl ester in medicaments for resisting the toxicity of staphylococcus aureus and biofilm formation, wherein the chemical structural formula of the Lo-tert-butyl ester is shown as a formula (1).
Further, the medicine is liquid, and the concentration of the Lo-tert-butyl ester in the medicine is not less than 25 mu M.
The invention also discloses application of the Lo-tert-butyl ester in preparing a medicament for treating diseases caused by staphylococcus aureus, wherein the chemical structural formula of the Lo-tert-butyl ester is shown as a formula (1).
The invention also discloses application of the Lo-tert-butyl ester in preparing a medicament for resisting the toxicity of staphylococcus aureus and biofilm formation, wherein the medicament comprises the Lo-tert-butyl ester, and the chemical structural formula of the Lo-tert-butyl ester is shown as a formula (1).
Further, the medicine is liquid, and the concentration of the Lo-tert-butyl ester in the medicine is not less than 25 mu M.
The invention also discloses application of the Lo-tert-butyl ester in preparing a disinfectant for preparing medical instruments or medical appliances, wherein the disinfectant comprises the Lo-tert-butyl ester, and the chemical structural formula of the Lo-tert-butyl ester is shown as the formula (1).
Further, the Lo-tert-butyl ester is prepared by adopting the following reaction route, namely, the reactant (I) desloratadine and the reactant (II) are subjected to amidation reaction, and then the Lo-tert-butyl ester is obtained by dehydration. Preferably, the reaction temperature is not lower than 150 ℃.
Further, the mole ratio of the reactant (I) desloratadine to the reactant (II) is 1: 1 to 1.5; wherein the reactant (II) is obtained by a conventional synthesis reaction.
Or the following reaction route is adopted, namely dissolving the reactant (I) desloratadine and the reactant (III) in an organic solvent, and reacting at-5-60 ℃ under the action of an acid binding agent.
Further, the organic solvent comprises one of dichloromethane, chloroform, acetone, acetonitrile, tetrahydrofuran, DMF, pyridine or toluene.
Further, the acid binding agent is at least one of triethylamine, arsenicum sablimatum, potassium tert-butoxide, sodium methoxide, sodium ethoxide, potassium carbonate, sodium carbonate, potassium bicarbonate, sodium hydroxide or potassium hydroxide.
Further, the mole ratio of the reactant (I) desloratadine to the reactant (III) is 1: 1 to 1.5; wherein the reactant (III) is obtained by a conventional synthesis reaction.
Although the compounds of the present invention may be administered directly without any formulation, the various compounds described are preferably used in the form of pharmaceutical preparations, the route of administration may be parenteral (e.g., intravenous, intramuscular) as well as oral.
Pharmaceutical compositions of the compounds of the invention are prepared as follows: the compounds of the present invention are combined with pharmaceutically acceptable solid or liquid carriers and optionally with pharmaceutically acceptable adjuvants and excipients using standard and conventional techniques to prepare microparticles or microspheres. Solid dosage forms include tablets, dispersible granules, capsules, sustained release tablets, sustained release pellets and the like. A solid carrier can be at least one substance that can act as a diluent, flavoring agent, solubilizing agent, lubricant, suspending agent, binder, disintegrant, and encapsulating agent. Inert solid carriers include magnesium phosphate, magnesium stearate, powdered sugar, lactose, pectin, propylene glycol, polysorbate 80, dextrin, starch, gelatin, cellulosic materials such as methylcellulose, microcrystalline cellulose, low melting waxes, polyethylene glycol, mannitol, cocoa butter, and the like. Liquid dosage forms include solvents, suspensions such as injections, powders, and the like.
Compared with the prior art, the invention has the beneficial effects that:
by adopting the technical scheme of the invention, a novel micromolecular compound Lo-tert-butyl ester with stronger inhibitory activity is obtained after modification and transformation are carried out on the side chain group of loratadine, and in vitro and in vivo experiments of animals prove that the micromolecular compound Lo-tert-butyl ester can effectively inhibit the toxicity and biofilm formation of staphylococcus aureus, does not influence the growth of bacteria, has no toxicity to mammalian cells and has no obvious hemolytic effect on human erythrocytes. The micromolecular compound Lo-tert-butyl ester can be used for preparing a medicament for inhibiting the virulence of staphylococcus aureus and the formation of a biofilm, or preparing a medicament for treating diseases caused by staphylococcus aureus, or preparing a disinfectant for medical instruments or medical appliances.
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FIG. 1 is a graph showing the inhibition of crystal violet staining of biofilm formation by strain HG003 of Staphylococcus aureus by Lo-tert-butyl ester in example 2 of the present invention.
FIG. 2 is a graph showing that Lo-tert-butyl ester of example 2 of the present invention inhibits the formation of biofilm of Staphylococcus aureus HG003 strain570And (5) detecting a result graph. Ratio of P to control<0.001(Student’s t test)。
FIG. 3 is a graph showing the analysis of growth of HG003 strain planktonic bacteria in example 2 of the present invention.
FIG. 4 is a graph showing the results of testing the inhibition of biofilm formation by Lo-tert-butyl ester of the 16 clinical strains of Staphylococcus aureus of example 2 of the present invention. Compared with the contrast ratio, the contrast ratio of the traditional Chinese medicine,*P<0.05；***P<0.001(Student’s t test)。
FIG. 5 is a graph showing the results of inhibition of pigment fading of 5 strains of Staphylococcus aureus by Lo-tert-butyl ester in example 3 of the present invention.
FIG. 6 shows that Lo-tert-butyl ester of example 3 of the present invention inhibits Staphylococcus aureus (OD) production from 5 strains of Staphylococcus aureus450Test) results analysis chart;**by comparison, P<0.01(Student’s t test)。
FIG. 7 shows that Lo-tert-butyl ester of example 3 of the present invention inhibits hemolytic activity (OD) of 5 strains of Staphylococcus aureus550Test) results analysis chart; comparison with，**P<0.01(Student’s t test)。
FIG. 8 is a graph of the results of the Lo-tert-butyl ester treatment of example 4 of the present invention to increase the survival rate of mice infected with the lung of strain HG003 of Staphylococcus aureus. Lo-tert-butyl ester (140mg/kg totally);*:P<0.01(Log-rank test)。
Detailed Description
Preferred embodiments of the present invention are described in further detail below.
Example 1
Lo-tert-butyl ester was prepared using the following procedure:
dissolving a reactant (I) desloratadine in an organic solvent, taking triethylamine as an acid-binding agent, and adding an organic solution in which a reactant (III) is dissolved, wherein the molar ratio of the reactant (I) desloratadine to the reactant (III) is 1: 1-1.5, and stirring the mixture at-5 ℃ for reaction to obtain a reaction solution containing Lo-tert-butyl ester. Wherein the organic solvent can be one of dichloromethane, trichloromethane, acetone, acetonitrile, tetrahydrofuran, DMF, pyridine or toluene. Wherein the reactant (III) is obtained by a conventional synthesis reaction.
After the reaction is finished, pouring the reaction solution into cold water, fully shaking for layering, extracting an organic layer, and continuously washing for three times. The organic layer was dried and left to stand. Then filtered, and the solvent was evaporated under reduced pressure to obtain the compound Lo-tert-butyl ester. The product was purified by silica gel column chromatography for subsequent experiments.
Example 2
Compound Lo-tert-butyl ester inhibits biofilm formation assay of staphylococcus aureus.
Detection of biofilm formation of strains: the staphylococcus aureus strain is cultured in a TSB culture medium at 37 ℃ and 220rpm/min overnight for 10-12 h. Bacterial suspension 1:200 (with or without Lo-t-butyl ester) was diluted with TSBG medium (TSB medium + 0.5% glucose), 96-well plates (Costar 3599) were added to 200. mu.l/well, 3 wells per strain were set, and the mixture was incubated at 37 ℃ for 24 hours. Discard the supernatantEluting with PBS 3 times (200 ul/well/time), drying at room temperature, adding methanol, fixing for 15min (200 ul/well), discarding methanol, drying at room temperature, adding 0.5% crystal violet dye solution 100ul per well, dyeing at room temperature for 10min, eluting the crystal violet dye solution with clear water until running water is colorless, drying at room temperature, reading OD on microplate reader570The value is obtained. The above experimental procedure was independently repeated 3 times, and the data were expressed as mean ± standard deviation (mean ± SD).
Detecting the growth of planktonic bacteria of the strain: culturing the strain in MHB culture medium at 37 deg.C and 220rpm/min overnight for 10-12h, diluting with fresh MHB culture medium 1:200 (with or without Lo-tert-butyl ester), culturing at 37 deg.C and 220rpm/min for 24h, and reading OD on microplate reader every 1h600The value is obtained.
As shown in figures 1 to 4, Lo-tert-butyl ester can remarkably inhibit the biofilm formation of staphylococcus aureus standard strain HG003 and 16 clinical isolates at the concentration of 25 mu M, but has no influence on the growth of planktonic bacteria.
Example 3
The compound Lo-tert-butyl ester is used for inhibiting the generation of staphylococcus aureus aureogenesis and hemolytic activity tests.
Detecting golden yellow pigment of the strain: after 48h incubation with TSB medium (with or without Lo-tert-butyl ester) at 37 deg.C, 3ml of the bacterial culture was centrifuged and washed twice with 0.01M Phosphate Buffered Saline (PBS). Washing the thallus sediment by PBS, discarding the supernatant, adding 300ul methanol (100%) into the thallus sediment, blowing and beating the resuspended bacteria liquid, shaking for 5 minutes after resuspension, centrifuging (12000r/min, centrifuging for 1-2 minutes), then sucking the supernatant extract into a clean EP tube, then adding 300 plus 350ul methanol (100%) into the thallus sediment, repeating for 2 times, sucking out the extract each time, finally fully mixing about 1ml of the 3 sucked-together extracts, then taking 200ul to a 96-well plate (3 multiple wells), and measuring the OD450 value on an enzyme labeling instrument. The above experimental procedure was independently repeated 3 times, and the data were expressed as mean ± standard deviation (mean ± SD).
And (3) detecting hemolytic activity of the strain: the strain 1:200 is inoculated in 4mL of TSB culture medium at 37 ℃ and 220rpm for 12h (activated bacteria), the activated bacteria 1:200 is inoculated in 6mL of TSB, the TSB is cultured at 37 ℃ and 220rpm for 12h (with or without Lo-tert-butyl ester), the strain is centrifuged at 4000rpm and 4 ℃ for 10min, supernatant is taken, bacteria in the supernatant culture solution are removed through a 0.22 mu m filter, and the supernatant culture solution is transferred to a sterile test tube for standby. Mu.l of each strain supernatant and 1% rabbit erythrocytes were mixed in 1.5ml EP tubes, TritonX-100 as positive control (100% hemolysis), 1 XPBS as negative control, incubated at 37 ℃ for 15min, centrifuged for 15min, 100. mu.l of supernatant was transferred to a new 96-well plate and the absorbance read at 550 nm. The above experimental procedure was independently repeated 3 times, and the data were expressed as mean ± standard deviation (mean ± SD).
As shown in FIGS. 5 to 7, Lo-tert-butyl ester (25. mu.M) significantly inhibited the formation of Staphylococcus aureus in 5 strains of Staphylococcus aureus, and significantly reduced the hemolytic activity of the strains.
Example 4
Test for improving survival rate of staphylococcus aureus lung infected mice treated by Lo-tert-butyl ester compound.
A C57BL/6J mouse Staphylococcus aureus lung infection model was constructed to evaluate the inhibitory effect of Lo-tert-butyl ester on Staphylococcus aureus virulence. The method comprises the following steps: 6-8 week-old C57BL/6J mice (18-20 g/mouse) were selected, 15 mice per group. Mice were anesthetized by intraperitoneal injection of 11.8mg/L sodium pentobarbital 100ul, 1 hour after anesthesia, Staphylococcus aureus HG003 strain (2.0X 10)9cfu) was titrated into nose, 20ul bacteria solution/mouse, then the experimental group mice started to inject Lo-tert-butyl ester into the abdominal cavity, 2 times in 1 day, each dose is 10mg/kg, and the duration is 1 week, and the accumulated total dose reaches 140 mg/kg. Mice were observed daily for survival. The above experimental procedures were independently repeated at least 2 times.
As shown in FIG. 8, the survival rate of mice infected with the lung of Staphylococcus aureus HG003 strain was significantly improved by treating with Lo-tert-butyl ester for 1 week (cumulative dose of 140 mg/kg).
Example 5
Inhibition of the growth of Staphylococcus aureus by the compound Lo-tert-butyl ester.
This example uses the standard tube dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI) of the United states:
1. the bacteria were inoculated into fresh MH liquid medium and cultured overnight at 37 ℃.
2. Correcting the concentration of the bacterial liquid to 0.5 McLeod turbidity standard by using a fresh MH liquid culture medium, and then adding the MH liquid culture medium according to the ratio of 1:200 dilution, 1mL of Lo-tert-butyl ester (final concentration of solvent DMSO is kept at 1%) with different concentration gradients, and incubation at 37 ℃ for 18 hours. Since Lo-tert-butyl ester was dissolved in DMSO, 0.1% DMSO + bacteria was used as a control and sterile medium was used as a blank.
3. And taking out and comparing with a blank control, wherein the tube with the lowest concentration in which bacteria do not grow is the minimum inhibitory concentration of the compound.
The results show that Lo-tert-butyl ester has no inhibitory effect on the growth of Staphylococcus aureus.
Example 6
The cytotoxicity test for detecting the compound Lo-tert-butyl ester by the MTT method comprises the following steps:
1. freshly cultured Vero cells were seeded into 96-well plates at 100. mu.L cells per well (approximately 5X 10)4Cells), 37 ℃, 5% CO2The cells were incubated for 24 hours under conditions to allow the cells to grow into a monolayer.
2. The medium was discarded, and 100. mu.L/well of fresh MEM medium containing different concentrations of the compound Lo-tert-butyl ester (the final concentration of the solvent DMSO was kept at 0.1%) was added to each sample in 6 wells, and the samples were loaded at 37 ℃ with 5% CO2The culture was continued under the conditions for 24 hours. Since Lo-tert-butyl ester was dissolved in DMSO, 0.1% DMSO + cells were used as a control.
3. mu.L of MTT marker was added to each well at 37 ℃ with 5% CO2Cultured under the conditions for 4 hours.
4. Adding 100 μ L of dissolving solution into each well, at 37 deg.C and 5% CO2Incubated under conditions overnight.
5. The 96-well plate was taken out to read the OD570Values, the average of 6 wells per sample reading, were calculated for the inhibition of Vero cell growth by compounds at different concentrations using the following formula:
half the inhibitory amount CC50The values were calculated using the Logit method. Table 1 shows the compound Lo-t-butylToxic effects of the ester on Vero cells.
TABLE 1
The results show that no toxicity to Vero cells was observed.
Example 7
An erythrocyte hemolysis experiment, comprising the following steps:
1. the isolated healthy human red blood cells were washed 3 times with sterile physiological saline and diluted to 5%.
2. Different concentrations of the small molecule compound Lo-tert-butyl ester (the final concentration of the solvent DMSO is kept at 1%) are added to a 5% erythrocyte suspension, 200. mu.l of each well is inoculated on a 96-well plate, and each sample adopts three-well. Because Lo-tert-butyl ester is dissolved in DMSO, 0.1% DMSO + cells are used as negative controls, 1% cell-permeable Triton-100+ cells are used as positive controls, and two common antibiotics, cefazolin and vancomycin, are used as controls. Incubate at 37 ℃ for 1 hour, centrifuge, and transfer 100. mu.l of the supernatant to another clean 96-well plate, OD570Readings, taking the mean of triplicate wells for each sample reading.
Table 2 shows the results of the toxic effect of Lo-tert-butyl ester compound on red blood cells.
TABLE 2 toxic Effect of Lo-tert-butyl ester compound on Red blood cells
The results show that: the small molecule compound Lo-tert-butyl ester has no hemolytic effect on human red blood cells.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (8)
1. An inhibitor Lo-tert-butyl ester against Staphylococcus aureus virulence and biofilm formation, characterized in that: the chemical structural formula is shown as formula (1):
2. a medicine for resisting staphylococcus aureus virulence and biofilm formation is characterized in that: which comprises the inhibitor Lo-tert-butyl ester against Staphylococcus aureus virulence and biofilm formation according to claim 1.
3. The agent of claim 2 for combating S.aureus virulence and biofilm formation, comprising: the medicine is liquid, and the concentration of the Lo-tert-butyl ester in the medicine is not less than 25 mu M.
Use of Lo-tert-butyl ester in a medicament against Staphylococcus aureus virulence and biofilm formation, characterized in that: the chemical structural formula of the Lo-tert-butyl ester is shown as the following formula (1):
5. use of Lo-tert-butyl ester according to claim 4 for the preparation of a medicament for the treatment of diseases caused by staphylococcus aureus, characterized in that: the medicine is liquid, and the concentration of the Lo-tert-butyl ester in the medicine is not less than 25 mu M.
Use of Lo-tert-butyl ester for the preparation of a medicament for the treatment of diseases caused by staphylococcus aureus, characterized in that: the chemical structural formula of the Lo-tert-butyl ester is shown as the following formula (1):
use of Lo-tert-butyl ester for the preparation of a medicament against Staphylococcus aureus virulence and biofilm formation, characterized in that: the medicine comprises Lo-tert-butyl ester, and the chemical structural formula of the Lo-tert-butyl ester is shown as the following formula (1):
use of Lo-tert-butyl ester for the preparation of a disinfecting liquid for the manufacture of medical instruments or medical devices, characterized in that: the disinfectant comprises Lo-tert-butyl ester, and the chemical structural formula of the Lo-tert-butyl ester is shown as the following formula (1):
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| NICHOLAS CUTRONA ET AL.: "From Antihistamine to Anti-infective: Loratadine Inhibition of Regulatory PASTA Kinases in Staphylococci Reduces Biofilm Formation and Potentiates β-Lactam Antibiotics and Vancomycin in Resistant Strains of Staphylococcus aureus", 《ACS INFECT. DIS.》 * |
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