Application of Etalocib in the preparation of drugs for treating Gram-positive bacterial infections

Abstract

The present invention provides an application of Etalocib in the preparation of an anti-Gram-positive bacterial infection drug, wherein the Etalocib has a CAS number of 161172-51-6; and the Etalocib has the function of inhibiting the growth of Gram-positive bacteria and the formation of biofilms. The technical solution of the present invention discloses a new medical use of Etalocib, wherein Etalocib exhibits antibacterial and antibacterial activity against a variety of Gram-positive bacteria, and can inhibit the formation of biofilms of Staphylococcus aureus and Enterococcus faecalis, can kill bacteria in mature biofilms of Staphylococcus aureus, and can significantly improve the survival rate of infected larvae in a wax moth larvae infection model.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to the use of Etalocib in preparing drugs for resisting Gram-positive bacteria infection. Background Art

[0002] In recent years, with the widespread use of antibiotics, the emergence and spread of multidrug-resistant (MDR) bacteria has become a major challenge in the field of global public health. The emergence and spread of these resistant bacteria have significantly accelerated, causing many common infections to become difficult to treat, posing a major challenge to the optimal antimicrobial treatment options in clinical practice, not only increasing the incidence rate, but also prolonging hospitalization time, leading to a significant increase in medical costs. What is more worrying is that the continuous evolution and accelerated development of antimicrobial resistance have seriously weakened the effectiveness of traditional antimicrobial drug treatment, making the treatment of bacterial infection-related diseases more difficult, and bringing higher medical risks and longer treatment cycles to patients.

[0003] In many cases of bacterial infection, more than 80% of the cases were found to form biofilms. This phenomenon greatly limits the diffusion and penetration of antimicrobial drugs and has become one of the main causes of chronic infection and antibiotic resistance. Biofilms are mainly composed of extracellular polymers (EPS), which are complex network structures containing components such as extracellular polysaccharides, proteins and extracellular DNA. This network structure not only provides a protective microenvironment for bacteria, but also morphologically resembles a barrier, allowing bacteria to adhere tightly inside the biofilm and jointly resist the attack of external antibiotics. Under the protection of the biofilm, bacteria can evade the recognition of the host immune system and the killing of antimicrobial drugs, making it difficult to effectively treat infections caused by related pathogens, often leading to the failure of clinical anti-infection treatment. Therefore, it is of great significance to develop new antibacterial infection drugs that can destroy biofilms and eliminate infections with drug-resistant strains. Summary of the invention

[0004] In view of the above technical problems, the present invention discloses the use of Etalocib in the preparation of an anti-Gram-positive bacterial infection drug. Etalocib exhibits antibacterial activity against clinical isolates of Gram-positive bacteria, can inhibit the formation of biofilms of Staphylococcus aureus and Enterococcus faecalis, and can kill bacteria in mature biofilms of Staphylococcus aureus. It can significantly improve the survival rate of infected larvae in a Galleria mellonella larvae infection model.

[0005] To this end, the technical solution adopted by the present invention is:

[0006] The invention discloses an application of Etalocib in preparing a drug for preventing Gram-positive bacteria infection. The CAS number of the Etalocib is 161172-51-6. The Etalocib has the function of inhibiting the growth of Gram-positive bacteria and the formation of biofilm.

[0007] Wherein, the structural formula of Etalocib is shown in formula (1):

[0008]

[0009] Leukotrienes are inflammatory mediators produced by the metabolism of arachidonic acid via the 5-lipoxygenase pathway. Etalocib is an orally active leukotriene (LTB4) receptor antagonist that exerts its anti-tumor effect by inhibiting the LTB4 receptor or directly targeting 5'-lipoxygenase, thereby inhibiting the leukotriene pathway. Etalocib not only inhibits the binding of [3H]LTB4, with a Ki value of 25nM, but also prevents LTB4-induced calcium mobilization, with an IC 50 The value is 20nM. Existing studies have shown that the leukotriene receptor inhibitor Etalocib can block the progression of leukotriene B4-induced lung inflammation, inhibit the proliferation of pancreatic cancer cells and promote cell apoptosis. In addition, the leukotriene receptor inhibitor Etalocib has a positive effect on the development of lung inflammation and promotes apoptosis of pancreatic cancer cells after intravenous injection (ED 50 =14 μg / kg) or oral (ED 50 =0.4 mg / kg), it can produce dose-related acute leukotriene B4-induced airway obstruction inhibition. So far, there is no report on the antibacterial activity of Etalocib compounds.

[0010] As a further improvement of the present invention, the Gram-positive bacteria is at least one of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis or Staphylococcus hominis.

[0011] Through a large number of experiments, it was found that the Etalocib compound showed antibacterial activity against a variety of Gram-positive bacteria, including clinical isolates of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium. The minimum inhibitory concentration (MIC) of Etalocib for 151 Gram-positive bacteria was 3.125-25μM. It is worth noting that the MIC for 39 clinically isolated methicillin-resistant Staphylococcus aureus (MRSA) was ≤25μM. Growth curve analysis showed that Etalocib could significantly delay the growth of Staphylococcus aureus and Enterococcus faecalis at a lower concentration (1 / 2×MIC). Bactericidal curve analysis showed that Etalocib could significantly kill Staphylococcus aureus at 2×MIC, and showed more significant bactericidal activity than vancomycin. Crystal violet staining showed that Etalocib could significantly inhibit the formation of biofilms of Staphylococcus aureus and Enterococcus faecalis at sub-inhibitory concentrations, and had strong killing activity against bacteria in biofilms of Staphylococcus aureus at a concentration of 4×MIC. Adding bacterial membrane phospholipid components to the culture medium can reduce the antibacterial activity of Etalocib compounds in a dose-dependent manner, indicating that Etalocib compounds exert their antibacterial activity by targeting bacterial cell membranes. In addition, Etalocib compounds have low toxicity to human cells, and their cytotoxicity to human liver cancer cells HepG2, human hepatic stellate cells LX-2, and human renal epithelial cell line 293T is higher than its minimum inhibitory concentration (MIC) value for bacteria. These results indicate that Etalocib compounds have potential application value in clinical anti-infection treatment.

[0012] As a further improvement of the present invention, the concentration of Etalocib in the treatment system is not less than 0.3906 μM. Further, the concentration of Etalocib compound in the treatment system is not less than 3.125 μM. Further, the concentration of Etalocib compound in the treatment system is 0.3906-200 μM.

[0013] As a further improvement of the present invention, the drug is a pharmaceutical composition or preparation. Further, the drug is an injection, tablet, pill, capsule, suspension, granule, spray or emulsion.

[0014] The present invention also discloses the use of Etalocib in preparing a coating for inhibiting Gram-positive bacteria. The coating is used on the surface of a medical device. The CAS number of the Etalocib is 161172-51-6, and the structural formula is shown in formula (1). The Etalocib has the function of inhibiting the growth of Gram-positive bacteria and the formation of biofilm.

[0015] As a further improvement of the present invention, in the coating, the concentration of Etalocib is not less than 0.3906 μM.

[0016] The present invention also discloses the use of Etalocib for preparing an anti-Gram-positive bacteria disinfectant. The CAS number of the Etalocib is 161172-51-6, and the structural formula is shown in formula (1). The disinfectant has good antibacterial or bactericidal effects on a variety of Gram-positive bacteria including Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis, Staphylococcus hominis, etc.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The technical scheme of the present invention discloses a new medical use of the Etalocib compound. The Etalocib compound exhibits antibacterial activity against clinical isolates of Gram-positive bacteria including Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis, and Staphylococcus hominis, and in particular, the inhibitory effect on Staphylococcus aureus is more obvious, and it exhibits more significant bactericidal activity than vancomycin; Etalocib can significantly inhibit the formation of biofilms of Staphylococcus aureus and Enterococcus faecalis at sub-inhibitory concentrations, and can kill bacteria in the biofilm of Staphylococcus aureus. In addition, the Etalocib compound has less toxicity to human liver cancer cells HepG2, human hepatic stellate cells LX-2, and human renal epithelial cell line 293T at the MIC dose, and is suitable for clinical use. These results suggest that the Etalocib compound has the possibility of treating a variety of bacterial infections. It can be seen that the Etalocib compound has potential application value in clinical anti-bacterial infection treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 3 is the growth curve of Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis after Etalocib in the embodiment of the present invention, in which A and B are the experimental results of methicillin-resistant Staphylococcus aureus (MRSA) YUSA139 and YUSA145, respectively; C and D are the experimental results of methicillin-sensitive Staphylococcus aureus (MSSA) SA113 and CHS101, respectively; E and F are the experimental results of clinical isolates of Enterococcus faecalis EF16C30 and EF16C396, respectively, and MIC in the figure represents the minimum inhibitory concentration.

[0020] Figure 21 is the bactericidal curve of Etalocib against Gram-positive bacteria Staphylococcus aureus in the embodiment of the present invention. In the figure, A is the experimental result of methicillin-sensitive Staphylococcus aureus (MSSA) clinical strain SA113; B is the experimental result of methicillin-resistant Staphylococcus aureus (MRSA) clinical strain YUSA145.

[0021] Figure 3 This is an analysis of the inhibition of biofilm formation of Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis by Etalocib in an embodiment of the present invention. In the figure, A is a crystal violet staining quantitative analysis of the effect of Etalocib on the biofilm formation of methicillin-sensitive Staphylococcus aureus (MSSA); B is a crystal violet staining quantitative analysis of the effect of Etalocib on the biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA); C is a crystal violet staining quantitative analysis of the effect of Etalocib on the biofilm formation of Enterococcus faecalis (E.faecalis), and MIC in the figure represents the minimum inhibitory concentration.

[0022] Figure 4 The results of the killing of bacteria in the biofilm of the clinical strain YUSA145 of Staphylococcus aureus by different concentrations of Etalocib (4×, 8× and 16×MIC) and different concentrations of Vancomycin (8× and 16×MIC) under laser confocal microscopy in an embodiment of the present invention are shown. Van represents vancomycin, and the scale bar in the figure is 16 μm.

[0023] Figure 5 3 and 4. The results of the effect of Etalocib on the cell membrane permeability of Staphylococcus aureus according to the embodiment of the present invention are shown in FIG. 3 . In the figure, A is a fluorescence intensity quantitative analysis of the effect of Etalocib on the cell membrane permeability of methicillin-sensitive Staphylococcus aureus (MSSA) SA113; B is a fluorescence intensity quantitative analysis of the effect of Etalocib on the cell membrane permeability of methicillin-resistant Staphylococcus aureus (MRSA) YUSA145.

[0024] Figure 6 This is the effect of adding different bacterial membrane phospholipid components on the antibacterial activity of Etalocib in the embodiment of the present invention. In the figure, CL represents cardiolipin, PG represents phosphatidylglycerol, and PE represents phosphatidylethanolamine.

[0025] Figure 7 This is the cytotoxicity test result of Etalocib in the embodiment of the present invention, wherein A is human liver cancer cell HepG2, B is human hepatic stellate cell LX-2, and C is human kidney epithelial cell line 293T.

[0026] Figure 8: is the anti-infection efficacy result of Etalocib of the embodiment of the present invention against Staphylococcus aureus infection, wherein A is the experimental scheme of Greater wax moth larvae infected with Staphylococcus aureus USA300; B is the survival rate of Greater wax moth larvae infected with Staphylococcus aureus USA300; C is a representative image of Greater wax moth larvae infected with Staphylococcus aureus USA300 and treated with Etalocib 48 hours later. DETAILED DESCRIPTION

[0027] The preferred embodiments of the present invention are described in further detail below.

[0028] Example 1

[0029] 151 Gram-positive bacteria (including 39 MRSA, 41 MSSA, 22 Staphylococcus epidermidis, 21 Enterococcus faecium, 19 Enterococcus faecalis and 9 Staphylococcus aureus) clinical strains used in the present embodiment were collected from different inpatients in the hospital. All clinical strains were identified by Phoenix 100 automatic microbial identification system, and matrix-assisted laser desorption ionization / time-of-flight mass spectrometry (MALDI-TOF-MS) was used to re-identify all strains after subculture. Quality control strain Staphylococcus aureus SA113 (ATCC35556) was purchased from ATCC strain library.

[0030] In this example, a 96-well plate high-throughput assay was used to detect the effect of Etalocib on the growth of Staphylococcus aureus and Enterococcus faecalis. The specific steps are:

[0031] Take the overnight culture solution (Staphylococcus aureus SA113 and Enterococcus faecalis OG1RF) and adjust the turbidity to 0.5 McFarland (the bacterial count is about 1.0-1.5×10 8 CFU / mL). The bacterial solution was diluted with CAMHB medium at 1:200 and added to a 96-well plate, with 12 wells in each row and 200μL in each well. The compounds in the custom compound library were added and diluted to 50μM, and 200μL CAMHB medium was added to the 12th well as a negative control. MRTX-1257, CTX-0294885, BAY1217389, N-Boc-O-tosylhydroxylamine, KPLH1130, MK2-IN-1 (hydrochloride), Ro 5126766, P110δ-IN-1, S55746, BI-3812 and other compounds were used as control samples. The results were observed after culturing at 37°C for 24 hours. The compounds added to the wells where bacterial growth could not be seen with the naked eye were considered to have potential antibacterial activity.

[0032] The experiment found that after 24 hours of adding 50 μM Etalocib to the culture medium of Staphylococcus aureus SA113 and Enterococcus faecalis OG1RF, the culture medium became clear and no bacterial growth was observed. 600 All of them were less than 0.1; other control compounds had no significant antibacterial activity. It can be seen that Etalocib has potential antibacterial activity against Staphylococcus aureus and Enterococcus faecalis.

[0033] Example 2

[0034] In this example, the minimum inhibitory concentration (MIC) of Etalocib against 151 Gram-positive bacteria (including 39 MRSA strains, 41 MSSA strains, 22 Staphylococcus epidermidis strains, 21 Enterococcus faecium strains, 19 Enterococcus faecalis strains and 9 Staphylococcus aureus strains) was detected by microbroth dilution method, and the specific steps included:

[0035] Take the overnight culture solution and adjust the turbidity to 0.5 McFarland (the bacterial count is about 1.0-1.5×10 8 CFU / mL). The bacterial solution was diluted with CAMHB medium at 1:100 and added to a 96-well plate, with 12 wells in each row. The drugs were set in 9-degree wells (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.7812, 0.3906 μM). 200 μL of the above bacterial solution was added to the 11th well as a positive control, and 200 μL of CAMHB medium was added to the 12th well as a negative control. The results were observed after culturing at 37°C for 18 hours, and the drug concentration well where the bacterial solution precipitation could not be seen by the naked eye was calculated as the MIC value.

[0036] In this embodiment, the statistical results of the MIC values ​​of Etalocib against Gram-positive bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Enterococcus faecalis and Enterococcus faecium are shown in Table 1. It can be seen that Etalocib has a relatively broad-spectrum antibacterial activity against a variety of Gram-positive bacteria, and the MIC values ​​are mainly distributed between 3.125 μM and 12.5 μM, among which the inhibitory effect on Enterococcus faecium is the best, with a MIC50 of 3.125 μM.

[0037] Table 1 Distribution of MIC values ​​of common Gram-positive bacteria to Etalocib

[0038]

[0039] Note: MIC: minimum inhibitory concentration; MSSA: methicillin-sensitive Staphylococcus aureus; MRSA: methicillin-resistant Staphylococcus aureus; S.epidermidis: Staphylococcus epidermidis; E.faecalis: Enterococcus faecalis; E.faecium: Enterococcus faecium; S.hominis: Staphylococcus hominis; n is the number of strains tested. MIC 50 MIC: drug concentration required to inhibit 50% growth of the tested bacteria; 90 : Drug concentration required to inhibit 90% growth of the test bacteria.

[0040] Example 3

[0041] Experiment on the effect of Etalocib on the growth of Gram-positive and Gram-negative bacteria.

[0042] In order to verify whether Etalocib can inhibit the growth of bacteria, in this example, four strains of Staphylococcus aureus (CHS101, SA113, YUSA139 and YUSA145) and two strains of Enterococcus faecalis (EF16C30 and EF16C396) were treated with different sub-inhibitory concentrations of Etalocib, and their absorbance at 600 nm (OD 600 ) value, the specific steps are:

[0043] The overnight culture was diluted 1000 times with tryptone soy broth (TSB) medium and added to a 96-well plate. Different concentrations (1 / 16×, 1 / 8×, 1 / 4×, 1 / 2× and 1×) of Etalocib were added and placed in an automatic growth curve analyzer. The absorbance at 600 nm (OD 600 ) absorbance value was used to detect the content of planktonic bacteria in the culture supernatant. The incubation temperature was 37°C and the growth curve was drawn.

[0044] Growth curve analysis Figure 1 As shown, it can be seen that at a concentration of 1 / 2×MIC, Etalocib inhibits the growth of all Staphylococcus aureus planktonic bacteria; when the concentration reaches 1×MIC, the growth of Staphylococcus aureus and Enterococcus faecalis is completely inhibited. These results preliminarily indicate that Etalocib has the potential to be used as an anti-bacterial infection drug.

[0045] Example 4

[0046] Effect of time and dose of etalocib on bactericidal activity against Gram-positive bacteria.

[0047] In this example, the time- and dose-dependent effects of the antibacterial activity of Etalocib on Gram-positive bacteria were studied and compared with the activity of the clinical antibiotic vancomycin. The specific steps included:

[0048] The logarithmic period (OD 600 =0.5) of MSSA clinical strain SA113 and MRSA clinical strain YUSA145 were diluted 100 times, and incubated with 1×, 2×, 4×MIC of Etalocib and 4×MIC of Vancomycin, respectively, on a shaker at 200 rpm. Subsequently, samples were collected at 0, 2, 6, and 24 hours, and continuous gradient dilutions were performed using sterile saline, and the samples were spread on TSB plates and incubated at 37°C, and the colony counts were counted after 24 hours. The colony counts were expressed in CFU / mL.

[0049] The sterilization curve analysis obtained is as follows Figure 2 As shown, it can be seen that Etalocib has a significant bactericidal effect on Staphylococcus aureus at 2×MIC, and the bacterial count is reduced to the detection limit within 6 hours. The bactericidal effect is stronger than that of 4×MIC vancomycin.

[0050] Example 5

[0051] In this example, the crystal violet method was used to study the biofilm-inhibiting activity of Etalocib against Enterococcus faecalis and Staphylococcus aureus strains. The specific steps were as follows:

[0052] The crystal violet staining method was used to detect the changes in the amount of biofilm, repeated 3 times, with 3 replicates each time, and the average value was taken as the final test result. The operation steps are briefly described as follows: take the overnight bacterial solution and dilute it 1000 times with TSBG medium containing 2% glucose in a 96-well plate, with 3 replicates in each group, add different sub-inhibitory concentrations of Etalocib, and use the blank medium without bacterial solution as the negative control, and the solvent DMSO as the positive control. After incubation in a 37°C incubator for 24 hours. Gently aspirate the culture medium, rinse with sterile PBS 3 times, and dry at room temperature. Fix with methanol for 15 minutes, stain with 1% crystal violet stain for 15 minutes, rinse with sterile water 3 times until the control well is colorless, and dry at room temperature. Add 200μL of anhydrous ethanol to each well to dissolve, shake for 1 minute, and measure the absorbance at 570nm in an enzyme reader.

[0053] Crystal violet staining results Figure 3 As shown, different sub-inhibitory concentrations of Etalocib have a significant inhibitory effect on the formation of biofilms of Enterococcus faecalis and Staphylococcus aureus; when Etalocib is 1 / 2×MIC, the biomass of biofilms formed by all Enterococcus faecalis and Staphylococcus aureus strains is significantly reduced, indicating that Etalocib can inhibit the formation of biofilms of Enterococcus faecalis and Staphylococcus aureus.

[0054] Example 6

[0055] This example uses fluorescence staining and laser confocal microscopy to study the activity of Etalocib against mature biofilm of Staphylococcus aureus strains. The specific steps are as follows:

[0056] Biofilm was constructed using glass-bottomed culture dishes, and the changes in live bacteria in the biofilm were observed using a laser confocal microscope. The steps were briefly described as follows: the bacterial solution of Staphylococcus aureus clinical MRSA strain YUSA145 cultured overnight was diluted 200 times with TSBG medium, 2 mL of the diluted bacterial solution was added to a 20 mm glass-bottomed culture dish, and the dish was incubated at 37°C for 24 hours to form a mature biofilm. The culture medium was aspirated, washed three times with sterile saline, and then TSBG medium containing different concentrations of Etalocib (4×, 8×, and 16×) was added to continue culturing for 24 hours. After washing with sterile PBS, Live / Dead BacLight TM Fluorescent dye, incubate at room temperature in dark for 30 minutes, LIVE / DEAD BacLight TM Fluorescent dyes include two different nucleic acid dyes that can quickly distinguish live bacteria with intact plasma membranes from dead bacteria with incomplete plasma membranes. Propidium iodide (PI) is used to detect membrane permeability. When it enters bacteria through a damaged cell membrane and binds to nucleic acids, the fluorescence intensity increases. It is then observed and photographed under a laser confocal microscope.

[0057] The biofilm was observed using a laser confocal microscope. Figure 4 As shown, it can be seen that when treated with 4×MIC of Etalocib, the number of live bacteria (green fluorescence) in the biofilm of Staphylococcus aureus strain was greatly reduced, and the proportion of dead bacteria (red fluorescence) increased. As the concentration increased, the proportion of dead bacteria (red fluorescence) further increased, indicating that Etalocib can have a strong killing effect on bacteria in the mature biofilm of Staphylococcus aureus.

[0058] Example 7

[0059] This example is a research experiment on the cell membrane permeability of Staphylococcus aureus YUSA145 by Etalocib, and the specific steps are as follows:

[0060] The logarithmic phase Gram-positive bacterial YUSA145 cells were adjusted to OD 600 =0.05, incubated with propidium iodide (PI) fluorescent dye in the dark. Then the suspension was treated with different concentrations of Etalocib (final concentrations of 1×, 2× and 4×MIC), with 0.1% DMSO as a control, and the fluorescence value of propidium iodide (PI) staining was monitored by a microplate reader after 1 hour.

[0061] The fluorescence intensity monitoring results are as follows Figure 5As shown, it was found that the proportion of red fluorescent bacteria in Staphylococcus aureus YUSA145 was significantly increased after Etalocib treatment, and the fluorescence value also increased in a gradient, indicating that the cell membrane permeability changed, indicating that Etalocib had a damaging effect on the cell membrane of Staphylococcus aureus.

[0062] Example 8

[0063] In this example, the antibacterial activity of Etalocib on the phospholipid components of bacterial cell membranes was studied. In order to analyze whether the antibacterial activity of Etalocib is related to the cell membrane, the checkerboard microbroth dilution method was used to detect the effects of different concentrations of bacterial cell membrane phospholipid components phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and cardiolipin (CL) on the minimum inhibitory concentration (MIC) of Etalocib. The specific steps are as follows:

[0064] Take the overnight culture solution of Staphylococcus aureus standard strain SA113 and MRSA clinical strain YUSA145 and adjust the turbidity to 0.5 McFarland (the bacterial count is about 1.0-1.5×10 8 CFU / mL). The bacterial solution was diluted with CAMHB medium at 1:100 and added to a 96-well plate, with 12 wells in each row. Eight gradient wells (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 μM) were set for Etalocib. 200 μL of the above bacterial solution was added to the 9th well as a positive control, and 200 μL of CAMHB medium was added to the 10th well as a negative control; 7 gradient wells (128, 64, 32, 16, 8, 4, 2 μg / mL) were set for cell membrane phospholipid components. The culture conditions and time for the determination of the MIC value of each antimicrobial drug were carried out in accordance with the CLSI guidelines. The results were observed after culturing at 37°C for 18 hours, and the drug concentration well at which the bacterial solution precipitation could not be seen by the naked eye was calculated as the MIC value.

[0065] The results were plotted with the concentration of different phospholipids as the horizontal axis and the MIC concentration fold change of Etalocib as the vertical axis. Figure 6 As shown, exogenous addition of bacterial cell membrane phospholipids, phosphatidylethanolamine (PE) in the culture medium had no effect on the antibacterial activity of Etalocib, while phosphatidylglycerol (PG) and cardiolipin (CL) reduced the antibacterial activity of Etalocib in a dose-dependent manner. This shows that Etalocib exerts its antibacterial activity by acting on the bacterial cell membrane.

[0066] Example 9

[0067] This example is an experiment on the effect of Etalocib on the cytotoxicity of human hepatoma cells HepG2, human hepatic stellate cells LX-2 and human renal epithelial cell line 293T. The specific steps are as follows:

[0068] Prepare 100 μL of human liver cancer cell HepG2, human hepatic stellate cell LX-2 and human renal epithelial cell line 293T cell suspension in a 96-well plate. Place the culture plate in an incubator for pre-culture for 24 hours (37°C, 5% CO2). Add 10 μL of different concentrations of Etalocib to the culture plate, and set up 8 gradient wells (100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 μM). Incubate the culture plate in an incubator for 24 hours, and add 10 μL of CCK-8 solution to each well. Incubate the culture plate in an incubator for 1-4 hours. Measure the absorbance at 450 nm with an enzyme reader. Then calculate the cell viability by absorbance.

[0069] The CCK-8 kit is a rapid and highly sensitive detection kit based on WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt) and is widely used in cell proliferation and cytotoxicity. Its working principle is: in the presence of electron coupling reagents, it can be reduced by dehydrogenases in mitochondria to generate highly water-soluble orange-yellow formazan products (formazan). The depth of color is proportional to cell proliferation and inversely proportional to cytotoxicity. The OD450 value is measured at a wavelength of 450nm using an enzyme reader to indirectly reflect cell activity. The results of etalocib's toxicity to HepG2, LX-2 and 293T cells are shown in the figure. Figure 7 As shown, it can be seen that Etalocib has low cytotoxicity, which is higher than its MIC value for most of the clinical strains of Gram-positive bacteria tested, and has potential clinical anti-infection application potential.

[0070] Example 10

[0071] This example is an in vivo anti-infection treatment experiment of Etalocib against Staphylococcus aureus USA300, and the specific steps are as follows:

[0072] The in vivo infection model of Staphylococcus aureus was established using the larvae of G. mellonella. To prepare the inoculum, Staphylococcus aureus USA300 was cultured in 5 mL of culture medium, incubated at 37°C for 20 h, and then diluted 1:100 and inoculated into 50 mL of fresh culture medium, and cultured at 37°C with shaking until the OD 600 The concentration of bacteria was about 0.5. The bacteria were collected by centrifugation for 10 minutes, resuspended in the same volume of 0.9% saline, partially diluted in multiples, and plated for counting. 50 μL of bacterial suspension (bacterial count was about 4×10 6CFU / mL) were injected subcutaneously. One hour after inoculation, drugs (doses of 5, 10 and 20 mg / kg) were directly applied subcutaneously to the infected area, and vancomycin (dosage of 20 mg / kg) was used as a control. The number of surviving larvae of G. mellonella was observed every 12 hours.

[0073] The results are as follows Figure 8 As shown, it can be seen that Etalocib at a dose of 5 mg / kg has a significant anti-infection effect on infections caused by Staphylococcus aureus USA300, and the effect is better than vancomycin.

[0074] All the above experiments were processed and plotted using GraphPad Prism 8.0 software. P ≤ 0.05 was considered statistically significant.

[0075] The above results show that the Etalocib compound exhibits good antibacterial activity against common clinical Gram-positive bacteria. Etalocib has a significant bactericidal effect on Staphylococcus aureus, and even at a concentration of 2×MIC, the bacterial count is reduced to the detection limit within 6 hours. In addition, Etalocib exhibits good anti-biofilm activity, can significantly inhibit the formation of biofilms of clinical strains of Staphylococcus aureus and Enterococcus faecalis, and has a strong killing effect on bacteria in mature biofilms of Staphylococcus aureus. Etalocib is less toxic to HepG2, LX-2 and 293T cells at the MIC dose. These results suggest that the Etalocib compound has the possibility of treating clinical bacterial infections. Moreover, the MIC of Etalocib is comparable to that of commonly used antibiotics in clinical practice, making it suitable for clinical use.

[0076] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. The use of Etalocib in the preparation of a drug for anti-Gram-positive bacterial infection, characterized in that: The Etalocib has a CAS number of 161172-51-6; the Gram-positive bacteria is at least one of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis or Staphylococcus hominis.

2. The use of Etalocib according to claim 1 for preparing a drug for anti-Gram-positive bacterial infection, characterized in that: The concentration of Etalocib in the treatment system is not less than 0.3906 μM.

3. The use of Etalocib according to claim 1 for preparing a drug for anti-Gram-positive bacterial infection, characterized in that: The medicine is in the form of injection, tablet, pill, capsule, suspension, granule, spray or emulsion.

4. The use of Etalocib in the preparation of a coating for inhibiting Gram-positive bacteria, characterized in that: The coating is used for the surface of medical devices, the CAS number of Etalocib is 161172-51-6, and the Gram-positive bacteria is at least one of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis or Staphylococcus hominis.

5. The use of Etalocib according to claim 4 for preparing a coating for inhibiting Gram-positive bacteria, characterized in that: In the coating, the concentration of Etalocib is not less than 0.3906 μM.

6. The use of Etalocib for preparing an anti-Gram-positive bacteria disinfectant, characterized in that: The CAS number of the Etalocib is 161172-51-6, and the Gram-positive bacteria is at least one of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium.

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