CN110818685A - A kind of inhibitor against Staphylococcus aureus virulence and biofilm formation Lo-isohexyl and use thereof - Google Patents

Abstract

The present invention provides Lo-isohexyl, an inhibitor against Staphylococcus aureus virulence and biofilm formation, and use thereof. The structural formula is shown in formula (1). The Lo-isohexyl, an inhibitor against Staphylococcus aureus virulence and biofilm formation of the present invention, has an inhibitory effect on Staphylococcus aureus virulence and biofilm formation, and can be used for the preparation of inhibiting Staphylococcus aureus virulence and biofilm formation Formed medicines, or preparation of medicines for the treatment of diseases caused by Staphylococcus aureus, or preparation of medical instruments or medical instrument disinfectants.

Description

A kind of inhibitor Lo-isohexyl and its use against Staphylococcus aureus virulence and biofilm formation

technical field

The invention belongs to the field of biotechnology, and in particular relates to Lo-isohexyl, an inhibitor against Staphylococcus aureus virulence and biofilm formation, and uses thereof.

Background technique

Staphylococcus aureus can infect different parts of the human body and cause a variety of infectious diseases, from common skin diseases such as folliculitis, acne, and stye, to deep and deadly pneumonia, endocarditis, osteomyelitis, and other metastatic complications. sexually transmitted diseases. During the process of Staphylococcus aureus invading the host and inducing host infection, it can secrete a variety of virulence factors such as hemolysin, extracellular protease, leukocidin and phenol-soluble protein, etc., so as to assist its invasion and damage to the host, and then induce disease. At present, with the widespread use of antibacterial drugs, the emergence of drug-resistant bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA), has brought difficulties to clinical treatment. Vancomycin and linezolid are very few antibacterial drugs that can treat MRSA infection, but more and more vancomycin-insensitive Staphylococcus aureus (VISA/hVISA) and linezolid have been found at home and abroad. The choice of clinical antibiotics is severely limited. In addition, Staphylococcus aureus can also adhere to the surface of human tissue cells or medical implant materials to form a biofilm structure composed of exopolysaccharide adhesion molecules, proteins, teichoic acid and extracellular DNA (eDNA), etc. Bacteria are susceptible to antimicrobial drugs and evade attack and phagocytosis by host immune cells, resulting in chronic infection and prolonged healing. A serious problem is that in recent years, with the wide application of medical implant materials such as various catheters, dialysis techniques, and prosthetic joints, nosocomial infections associated with Staphylococcus aureus biofilms are increasing day by day. Current studies have found that drug-resistant strains of Staphylococcus aureus such as MRSA also have strong virulence, which can cause patients to develop severe infectious diseases such as septic shock and endocarditis, resulting in a high mortality rate. The strains also have strong biofilm forming ability. Therefore, in order to reduce the death of patients caused by these strains of infection, inhibiting the virulence and biofilm formation of Staphylococcus aureus has become one of the difficult and hot issues to be solved urgently in recent years.

SUMMARY OF THE INVENTION

In view of the above technical problems, the present invention discloses Lo-isohexyl, an inhibitor against Staphylococcus aureus virulence and biofilm formation, and uses thereof, which have the functions of inhibiting Staphylococcus aureus virulence and biofilm formation, and are effective against Staphylococcus aureus virulence and biofilm formation. Mammalian cells are not significantly toxic.

To this, the technical scheme adopted in the present invention is:

A kind of inhibitor Lo-isohexyl for anti-Staphylococcus aureus virulence and biofilm formation, its chemical structural formula is as follows:

Among them, Lo-isohexyl, whose molecular formula is C ₂₆ H ₃₁ ClN ₂ O ₂ , is a colorless, odorless and transparent liquid at room temperature, with high boiling point, good thermal stability and aprotic properties, and can be dissolved in ethanol and propanol , benzene and chloroform and most organic compounds. The Lo-isohexyl group is a small molecule compound. The small molecule compound Lo-isohexyl can obviously inhibit the virulence and biofilm formation of Staphylococcus aureus in vitro and in vivo; the small molecule compound Lo-isohexyl does not affect the growth of bacteria; it has no obvious toxicity to mammalian cells, and No significant hemolysis on human erythrocytes.

In the present invention, the described small molecular compound Lo-isohexyl can be used to be formulated into medical equipment or medical equipment disinfectant, and can further carry out the transformation of chemical structure to prepare a new drug against gram-positive bacteria infection.

The present invention also discloses a medicine against Staphylococcus aureus virulence and biofilm formation, which comprises Lo-isohexyl, an inhibitor against Staphylococcus aureus virulence and biofilm formation as described above.

Further, the medicine is liquid, and the concentration of the Lo-isohexyl group in the medicine is not less than 25 μM.

The present invention also discloses the use of Lo-isohexyl in a drug against Staphylococcus aureus virulence and biofilm formation, and the chemical structural formula of the Lo-isohexyl is shown in formula (1).

Further, the medicine is liquid, and the concentration of the Lo-isohexyl group in the medicine is not less than 25 μM.

The present invention also discloses the use of Lo-isohexyl in preparing a medicine for treating diseases caused by Staphylococcus aureus, and the chemical structural formula of the Lo-isohexyl is shown in formula (1).

The present invention also discloses the application of Lo-isohexyl in the preparation of medicines against Staphylococcus aureus virulence and biofilm formation, the medicine comprises Lo-isohexyl, and the chemical structural formula of the Lo-isohexyl is as follows 1) shown.

Further, the medicine is liquid, and the concentration of the Lo-isohexyl group in the medicine is not less than 25 μM.

The invention also discloses the application of Lo-isohexyl for preparing a disinfectant for medical equipment or medical equipment, the disinfectant includes Lo-isohexyl, and the chemical structural formula of the Lo-isohexyl is as shown in formula (1). Show.

Further, the Lo-isohexyl group is prepared by the following reaction route, that is, the desloratadine reactant (I) and the reactant (II) are subjected to amidation reaction, and the Lo-isohexyl group is obtained by dehydration. Preferably, the reaction temperature is not lower than 150 degrees.

Further, the molar ratio of desloratadine reactant (I) to reactant (II) is 1:1-1.5. wherein the reactant (II) is obtained by a conventional synthetic reaction.

Alternatively, the following reaction route is adopted, namely, the desloratadine reactant (I) and the reactant (III) are dissolved in an organic solvent, and the reaction is carried out at -5 to 60 degrees under the action of an acid binding agent.

Further, the organic solvent includes one of dichloromethane, chloroform, acetone, acetonitrile, tetrahydrofuran, DMF, pyridine or toluene.

Further, the acid binding agent includes triethylamine, arsenic, potassium tert-butoxide, sodium methoxide, sodium ethoxide, potassium carbonate, sodium carbonate, potassium bicarbonate, sodium bicarbonate, sodium hydroxide or potassium hydroxide. at least one of.

Further, the molar ratio of desloratadine reactant (I) to reactant (III) is 1:1-1.5. Wherein the reactant (III) is obtained by a conventional synthetic 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 formulations, which may be administered parenterally (eg, intravenously, intramuscularly) and orally.

Pharmaceutical compositions of the compounds of the present invention are prepared by combining the compounds of the present invention with a pharmaceutically acceptable solid or liquid carrier, and optionally with pharmaceutically acceptable auxiliaries and excipients, using standard and conventional techniques The formulations are combined into 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 which can act as a diluent, flavoring agent, solubilizer, lubricant, suspending agent, binder, disintegrating agent, and encapsulating agent. Inert solid carriers include magnesium phosphate, magnesium stearate, sugar syrup, lactose, pectin, propylene glycol, polysorbate 80, dextrin, starch, gelatin, cellulosics such as methylcellulose, microcrystalline cellulose, Low melting point paraffin, polyethylene glycol, mannitol, cocoa butter, etc. Liquid dosage forms include solvents, suspensions such as injections, powders, and the like.

Compared with the prior art, the beneficial effects of the present invention are:

By adopting the technical scheme of the present invention, after modifying the side chain group of loratadine, a novel small molecule compound Lo-isohexyl with stronger inhibitory activity is obtained. The compound Lo-isohexyl can effectively inhibit the virulence and biofilm formation of Staphylococcus aureus, and does not affect the growth of bacteria, has no toxicity to mammalian cells, and has no obvious hemolysis to human erythrocytes. The small molecular compound Lo-isohexyl can be used to prepare medicines for inhibiting the virulence and biofilm formation of Staphylococcus aureus, or for preparing medicines for treating diseases caused by Staphylococcus aureus, or for making medical instruments or medical instrument disinfectants.

Description of drawings

Fig. 1 is the crystal violet staining diagram of Lo-isohexyl inhibiting the biofilm formation of Staphylococcus aureus HG003 strain in Example 2 of the present invention.

Fig. 2 is a graph showing the results of OD ₅₇₀ detection of Lo-isohexyl inhibiting the formation of a biofilm of Staphylococcus aureus HG003 strain in Example 2 of the present invention. ***P<0.001 compared to control (Student's t test).

Fig. 3 is the growth detection and analysis diagram of HG003 strain planktonic bacteria in Example 2 of the present invention.

4 is a graph showing the detection results of Lo-isohexyl inhibiting the biofilm formation of 16 clinical strains of Staphylococcus aureus in Example 2 of the present invention. ^* P<0.05; ^** P<0.01; ^*** P<0.001 (Student's t test) vs. control.

Fig. 5 is a graph showing the results of the inhibition of pigment reduction of 5 strains of Staphylococcus aureus by Lo-isohexyl in Example 3 of the present invention.

Figure 6 is an analysis diagram of the results of Lo-isohexyl inhibiting the production of golden yellow pigment (OD ₄₅₀ detection) of 5 strains of Staphylococcus aureus in Example 3 of the present invention; ^** Compared with the control, P<0.01 (Student's t test).

Fig. 7 is the result analysis diagram of Lo-isohexyl inhibiting the hemolytic activity (OD ₅₅₀ detection) of 5 strains of Staphylococcus aureus in Example 3 of the present invention; compared with the control, ^* P<0.05; ^** P<0.01 (Student's t test).

Fig. 8 is an analysis diagram of the result that Lo-isohexyl treatment in Example 4 of the present invention improves the survival rate of mice infected with Staphylococcus aureus HG003 strain. Lo-isohexyl (140mg/kg totally);^*:P<0.01(Log-rank test).

Detailed ways

The preferred embodiments of the present invention will be further described in detail below.

Example 1

Lo-isohexyl was prepared using the following steps:

The desloratadine reactant (I) is dissolved in an organic solvent, and triethylamine is used as an acid binding agent, and an organic solution dissolved with reactant (III) is added, wherein reactant (I) and reactant (III) are added. ) in a molar ratio of 1:1 to 1.5, the two were mixed, and the reaction was stirred at -5°C to obtain a reaction solution containing Lo-isohexyl. The organic solvent can be one of methylene chloride, chloroform, acetone, acetonitrile, tetrahydrofuran, DMF, pyridine or toluene. Wherein reactant (III) is obtained by conventional synthetic reaction.

After the reaction, the reaction solution was poured into cold water, fully shaken for layers, the organic layer was extracted, and washed with water three times in a row. The organic layer was dried and left to stand. Then, it was filtered, and the solvent was evaporated under reduced pressure to obtain the compound Lo-isohexyl. The obtained product was purified by silica gel column chromatography and used in subsequent experiments.

Example 2

The compound Lo-isohexyl inhibits the biofilm formation of Staphylococcus aureus.

Strain biofilm formation detection: Staphylococcus aureus strains were cultured overnight in TSB medium at 37° C., 220 rpm/min shaking for 10-12 hours. Dilute the bacterial solution 1:200 with TSBG medium (TSB medium + 0.5% glucose) (with or without Lo-isohexyl), add 200ul per well to a 96-well plate (Costar 3599), and set 3 replicate wells for each strain , 37 ℃ static culture for 24h. Discard the supernatant, wash 3 times with PBS (200ul/well/time), add methanol to fix for 15min (200ul/well) after drying at room temperature, discard methanol and dry at room temperature and add 100ul of 0.5% crystal violet staining solution to each well, Stain at room temperature for 10 minutes, gently wash out the crystal violet staining solution under water until the running water is colorless, and read the OD ₅₇₀ value on a microplate reader after drying at room temperature. The above experimental operations were repeated 3 times independently, and the data were expressed as mean±standard deviation (mean±SD).

Phytoplankton growth detection of strains: After the strains were incubated overnight in MHB medium at 37°C and 220rpm/min for 10-12h, they were diluted 1:200 with fresh MHB medium (with or without Lo-isohexyl) at 37°C. , 220rpm/min culture for 24h, read the OD ₆₀₀ value on the microplate reader every 1h.

As shown in Figures 1 to 4, Lo-isohexyl at a concentration of 25 μM could significantly inhibit the formation of biofilms of the standard S. aureus strain HG003 and 16 clinical isolates, but had no effect on the growth of planktonic bacteria.

Example 3

Compound Lo-isohexyl inhibits Staphylococcus aureus production and hemolytic activity.

Strain golden yellow pigment detection: Incubate with TSB medium for 48h at 37°C (with or without Lo-isohexyl), centrifuge 3ml bacterial culture and wash twice with 0.01M phosphate buffered saline (PBS). After washing the cell pellet with PBS, discard the supernatant, add 300ul methanol (100%) to the cell pellet, resuspend the bacterial solution by pipetting, shake for 5 minutes after resuspending, centrifuge (12000r/min, centrifuge for 1-2 minutes), and then Aspirate the supernatant extract into a clean EP tube, then add 300-350ul methanol (100%) to the cell pellet, repeat 2 times, suck out the extract each time, and finally extract the 3 times together. About 1ml of the solution was mixed well, and 200ul was added to a 96-well plate (3 duplicate wells), and the OD450 value was measured on a microplate reader. The above experimental operations were repeated 3 times independently, and the data were expressed as mean±standard deviation (mean±SD).

Detection of hemolytic activity of strains: strains were inoculated at 1:200 in 4mL TSB medium at 37°C, 220rpm, and cultured for 12h (activated bacteria); after activation, bacteria were inoculated into 6ml TSB at 1:200, at 37°C, and cultured at 220rpm for 12h (with or without Lo -Isohexyl), centrifuge at 4000rpm for 10min at 4°C, take the supernatant, remove the bacteria in the supernatant culture medium through a 0.22 μm filter, and transfer it to a sterile test tube for later use. The strain supernatant and 1% rabbit erythrocytes were mixed with 500 μl each in a 1.5ml EP tube, TritonX-100 was used as a positive control (100% hemolysis), 1×PBS was used as a negative control, incubated at 37°C for 15min, centrifuged for 15min, and the 100 μl of the supernatant was transferred to a new 96-well plate and the absorbance was read at 550 nm. The above experimental operations were repeated 3 times independently, and the data were expressed as mean±standard deviation (mean±SD).

As shown in Figures 5 to 7, Lo-isohexyl (25 µM) can significantly inhibit the production of aureus pigment in 5 strains of Staphylococcus aureus, and significantly reduce the hemolytic activity of the strains.

Example 4

Compound Lo-isohexyl treatment improves survival in mice with Staphylococcus aureus pulmonary infection.

A C57BL/6J mouse model of S. aureus lung infection was constructed to evaluate the inhibitory effect of Lo-isohexyl on S. aureus virulence. The method is as follows: 6-8 week old C57BL/6J mice (18-20g/mice) were selected, 15 mice in each group. Mice were anesthetized by intraperitoneal injection of 11.8mg/L pentobarbital sodium 100ul, 1 hour after anesthesia, intranasal instillation of Staphylococcus aureus HG003 strain ( ^2.0x109 cfu), 20ul bacterial solution/mice, and then mice in the experimental group Begin intraperitoneal injection of Lo-isohexyl, 2 times a day, each dose of 10 mg/kg for 1 week, and the cumulative 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 Figure 8, treatment with Lo-isohexyl for 1 week (cumulative dose of 140 mg/kg) can significantly improve the survival rate of mice infected with Staphylococcus aureus HG003 strain.

Example 5

Inhibitory experiment of compound Lo-isohexyl on the growth of Staphylococcus aureus.

This example uses the standard test tube dilution method recommended by U.S. Clinical and Laboratory Standards Institute (CLSI):

1. Inoculate the bacteria in fresh MH liquid medium and cultivate overnight at 37°C.

2. Correct the bacterial concentration to 0.5 McFarland turbidity standard with fresh MH liquid medium, then dilute it with MH liquid medium at 1:200, add 1 mL to each test tube, and add 1 mL of different concentration gradients. of Lo-isohexyl (solvent DMSO at a final concentration of 1%), incubated at 37°C for 18 hours. Because Lo-isohexyl was dissolved in DMSO, 0.1% DMSO + bacteria was used as a control, and sterile medium was used as a blank control.

3. Take out the tube with the lowest concentration of bacteria that does not grow compared with the blank control, which is the minimum inhibitory concentration of the compound.

The results showed that Lo-isohexyl had no inhibitory effect on the growth of Staphylococcus aureus.

Example 6

The cytotoxicity test of compound Lo-isohexyl by MTT method includes the following steps:

1. Inoculate freshly cultured Vero cells in a 96-well plate, 100 μL of cells (about 5×10 ⁴ cells) per well, and culture at 37° C. and 5% CO ₂ for 24 hours to grow the cells into a monolayer.

2. Discard the medium and add 100 μL/well of fresh MEM medium containing different concentrations of compound Lo-isohexyl (the final concentration of solvent DMSO is kept at 0.1%), and each sample is loaded in 6 duplicate wells at 37°C , 5% CO ₂ for 24 hours. Since Lo-isohexyl was dissolved in DMSO, 0.1% DMSO+ cells were used as a control.

3. Add 10 μL of MTT marker to each well and incubate for 4 hours at 37°C and 5% CO ₂ .

4. Add 100 μL of lysate to each well and incubate overnight at 37°C and 5% CO ₂ .

5. Take out the 96-well plate and read the OD ₅₇₀ value, take the average value of 6 duplicate wells for each sample reading, and calculate the inhibition rate of the compound of different concentrations on the growth of Vero cells:

The _half -inhibition CC50 value was calculated by the Logit method. Table 1 shows the toxic effects of compound Lo-isohexyl on Vero cells.

Table 1

The results showed that no toxicity to Vero cells was observed.

Example 7

The red blood cell hemolysis test includes the following steps:

1. The isolated healthy human red blood cells were washed 3 times with sterile normal saline and diluted to 5%.

2. Add different concentrations of small molecule compound Lo-isohexyl (solvent DMSO final concentration is kept at 1%) in 5% red blood cell suspension, 200 μl per well are inoculated on 96-well plate, and each sample adopts three replicate wells. Because Lo-isohexyl was dissolved in DMSO, 0.1% DMSO+ cells were used as negative control, 1% cell penetrant Triton-100+ cells were used as positive control, and two commonly used antibiotics, cefazolin and vancomycin, were used. control. Incubate in a 37°C incubator for 1 hour. After centrifugation, 100 μl of the supernatant was taken and transferred to another clean 96-well plate. The OD ₅₇₀ was read, and the average of three replicate wells was taken for each sample reading.

Table 2 shows the results of the toxic effect of compound Lo-isohexyl on red blood cells.

Table 2 Toxicity of compound Lo-isohexyl to red blood cells

The results showed that the small molecule compound Lo-isohexyl had no hemolytic effect on human erythrocytes.

The above content is a further detailed description of the present invention in combination with specific preferred embodiments, and it cannot be considered that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the technical field of the present invention, without departing from the concept of the present invention, some simple deductions or substitutions can be made, which should be regarded as belonging to the protection scope of the present invention.

Claims (8)

1. the inhibitor Lo-isohexyl that resists Staphylococcus aureus virulence and biofilm formation is characterized in that: its chemical structural formula is shown in formula (1):

2. A drug against Staphylococcus aureus virulence and biofilm formation, characterized in that: it comprises Lo-isohexyl, an inhibitor against Staphylococcus aureus virulence and biofilm formation as claimed in claim 1. 3. The medicine against Staphylococcus aureus virulence and biofilm formation according to claim 2, wherein the medicine is a liquid, and the concentration of the Lo-isohexyl group in the medicine is not less than 25 μM. 4. the purposes of Lo-isohexyl in the medicine of anti-Staphylococcus aureus virulence and biofilm formation, it is characterized in that: the chemical structural formula of described Lo-isohexyl is shown in the following formula (1):

5. the purposes of the Lo-isohexyl group according to claim 4 for the preparation of the medicine for the treatment of the disease caused by Staphylococcus aureus, it is characterized in that: the medicine is a liquid, and in the medicine, the Lo-isohexyl group The concentration is not less than 25 μM. 6. the purposes of Lo-isohexyl for the preparation of the medicine for the treatment of diseases caused by Staphylococcus aureus, it is characterized in that: the chemical structural formula of described Lo-isohexyl is shown in the following formula (1):

7. the application of Lo-isohexyl in the medicine for preparing anti-Staphylococcus aureus virulence and biofilm formation, it is characterized in that: described medicine comprises Lo-isohexyl, and the chemical structural formula of described Lo-isohexyl is as follows (1) shows:

8. Lo-isohexyl is used to prepare the application of the sterilizing solution made into medical equipment or medical appliance, it is characterized in that: described sterilizing solution comprises Lo-isohexyl, and the chemical structural formula of described Lo-isohexyl is as follows (1) shown:

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