CN110698457B - 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 PDF

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CN110698457B
CN110698457B CN201910949487.0A CN201910949487A CN110698457B CN 110698457 B CN110698457 B CN 110698457B CN 201910949487 A CN201910949487 A CN 201910949487A CN 110698457 B CN110698457 B CN 110698457B
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郑金鑫
余治健
邓启文
尚永朋
林志伟
陈重
孙翔
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SHENZHEN NANSHAN DISTRICT PEOPLE'S HOSPITAL
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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

Inhibitor Lo-tert-butyl ester for resisting staphylococcus aureus virulence and biofilm formation and application thereof
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 resistant strains are found, so that the selection of clinical antibacterial drugs is severely limited. In addition, the 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 protraction 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 the 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:
Figure BDA0002225316290000021
wherein Lo-tert-butyl ester has a molecular formula of C 27 H 31 CIN 2 O 4 The 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 on human red blood cells.
In the invention, the micromolecular compound Lo-tert-butyl ester can be used for preparing medical instruments or medical appliance disinfectant, and can be further used for modifying chemical structures to prepare novel medicaments for resisting gram-positive bacteria infection.
The invention also discloses a medicament for resisting the toxicity and biofilm formation of the staphylococcus aureus, which comprises the inhibitor Lo-tert-butyl ester for resisting the toxicity and biofilm formation of the staphylococcus aureus.
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 ℃.
Figure BDA0002225316290000031
Further, the molar 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 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.
Figure BDA0002225316290000032
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 molar ratio of the reactant (I) desloratadine to the reactant (III) is 1:1 to 1.5; wherein the reactant (III) is obtained by 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 being parenteral (e.g. intravenous, intramuscular) as well as oral.
Pharmaceutical compositions of the compounds of the invention were 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, disintegrating agent, 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 small molecular compound Lo-tert-butyl ester can be used for preparing a medicament for inhibiting the toxicity and biofilm formation of staphylococcus aureus, or preparing a medicament for treating diseases caused by staphylococcus aureus, or preparing a medical instrument or medical appliance disinfectant.
Drawings
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 strain HG003 of Staphylococcus aureus 570 And (5) detecting a result graph. * Comparison of P with 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 aureus 450 Test) 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 Staphylococcus aureus strain 5 550 Test) results analysis chart; compared with the contrast ratio, the contrast ratio of the traditional Chinese medicine, ** 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 (140 mg/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:
Figure BDA0002225316290000051
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 to 1.5, and stirring the mixture at the temperature of minus 5 ℃ for reaction to obtain reaction liquid 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 10-12h. Bacterial suspension 1 was diluted with TSBG medium (TSB medium +0.5% glucose) in 200ul (with or without Lo-t-butyl ester) per well in 96-well plates (Costar 3599) with 3 wells per bacterial strain, and incubated at 37 ℃ for 24h. Discarding supernatant, eluting with PBS for 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, and reading OD on microplate reader 570 The 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: after overnight incubation of the strain in MHB medium at 37 ℃ and 220rpm/min for 10-12h, the strain was diluted with fresh MHB medium 1 (with or without Lo-t-butyl ester), incubated at 37 ℃ and 220rpm/min for 24h, and the OD read on a microplate reader every 1h 600 The 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-t-butyl ester) at 37 deg.C, 3ml of the bacterial culture was centrifuged and washed twice with 0.01M Phosphate Buffered Saline (PBS). After the bacterial precipitation is washed by PBS, the supernatant is discarded, 300ul of methanol (100%) is added into the bacterial precipitation, the resuspended bacterial liquid is blown and beaten, the cell is shaken for 5 minutes after the resuspension, the cell is centrifuged (12000 r/min and 1-2 minutes), then the supernatant extract is sucked into a clean EP tube, then 300-350ul of methanol (100%) is added into the bacterial precipitation, the operation is repeated for 2 times, the extract is sucked out every time, finally about 1ml of the extract sucked together for 3 times is fully mixed, 200ul of the extract is added into a 96-well plate (3-well), and the OD450 value is measured 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: strain 1. 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 is constructed to evaluate the inhibition effect of Lo-tert-butyl ester on the toxicity of staphylococcus aureus. 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, and 1 hour after anesthesia, staphylococcus aureus HG003 strain (2.0x10) 9 cfu) for nasal drip, 20ul bacteria solution/mouse, then experimental group mice started to inject Lo-tert-butyl ester intraperitoneally for 1 day 2 times, each dose was 10mg/kg for 1 week, and the cumulative total dose reached 140mg/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. Lo-tert-butyl ester was dissolved in DMSO, and 0.1% DMSO + bacteria was used as a control, and a 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) 4 Cells), 37 ℃,5% CO 2 The cells were cultured for 24 hours under conditions to grow 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 maintained at 0.1%) was added to each sample by 6-well loading, 37 ℃ and 5% CO 2 The 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. Add 10. Mu.L of MTT marker per well, 37 ℃ C., 5% CO 2 Cultured under the conditions for 4 hours.
4. Adding 100 μ L of dissolving solution per well, 37 deg.C, 5% 2 Incubated under conditions overnight.
5. The 96-well plate was taken out to read the OD 570 Values, 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:
Figure BDA0002225316290000081
half inhibition amount CC 50 The values were calculated using the Logit method. Table 1 shows the toxic effect of Lo-tert-butyl ester compound on Vero cells.
TABLE 1
Figure BDA0002225316290000082
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. Adding small molecules of different concentrationsThe compound Lo-tert-butyl ester (final concentration of solvent DMSO is kept at 1%) was seeded in a 96-well plate in 200. Mu.l/well in a 5% erythrocyte suspension, and triplicate wells were used for each sample. Since Lo-tert-butyl ester was dissolved in DMSO, 0.1% DMSO + cells were used as a negative control, 1% cell-permeable Triton-100+ cells were used as a positive control, and two common antibiotics, cefazolin and vancomycin, were used as controls. Incubate at 37 ℃ for 1 hour, centrifuge, and transfer 100. Mu.l of the supernatant to another clean 96-well plate, OD 570 Readings, 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
Figure BDA0002225316290000091
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 further detailed description of the invention in connection with specific preferred embodiments and it is not intended to limit the invention to the specific embodiments described. For those skilled in the art to which the invention pertains, numerous simple deductions or substitutions may be made without departing from the spirit of the invention, which shall be deemed to belong to the scope of the invention.

Claims (7)

1. An inhibitor Lo-tert-butyl ester for inhibiting virulence and biofilm formation of Staphylococcus aureus, comprising: the chemical structural formula is shown as formula (1):
Figure FDA0004080073100000011
2. a medicament for inhibiting staphylococcus aureus virulence and biofilm formation, comprising: which comprises the inhibitor Lo-tert-butyl ester of claim 1 for inhibiting virulence and biofilm formation of Staphylococcus aureus.
3. The medicament for inhibiting staphylococcus aureus virulence and biofilm formation according to claim 2, wherein:
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 inhibiting 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):
Figure FDA0004080073100000012
5. use of Lo-tert-butyl ester according to claim 4 for the preparation of a medicament for inhibiting S.aureus virulence and biofilm formation, 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 a disease caused by staphylococcus aureus, characterized in that: the chemical structural formula of the Lo-tert-butyl ester is shown as the following formula (1):
Figure FDA0004080073100000021
use of lo-tert-butyl ester for the preparation of a disinfecting liquid for medical instruments or medical devices, characterized in that: the chemical structural formula of the Lo-tert-butyl ester is shown as the following formula (1):
Figure FDA0004080073100000022
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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;Nicholas Cutrona et al.;《ACS Infect. Dis.》;20190527;摘要,第1406页第2段 *

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