Use of PCC0208009 in the manufacture of a medicament for the treatment of an infection by Staphylococcus aureus and Enterococcus spp.

Abstract

The application discloses a use of PCC0208009 in preparation of a drug for resisting staphylococcus aureus and enterococcus infection, wherein the PCC0208009 has a CAS number of 1668565-74-9; and the enterococcus is at least one of enterococcus faecalis and enterococcus faecium. Experiments show that the PCC0208009 has significant bacteriostatic activity on clinically common drug-resistant bacteria such as staphylococcus aureus, enterococcus faecalis and enterococcus faecium, and can significantly inhibit the formation of staphylococcus aureus biofilm; bacterial membrane physiology and electron microscope observation results show that the PCC0208009 may play an antibacterial activity by destroying the integrity of bacteria; cell proliferation experiment results show that there is no cytotoxicity in the antibacterial MIC concentration range, which indicates that the PCC0208009 has a broad clinical application prospect in preparation of a drug for treating gram-positive bacteria (especially drug-resistant strains and biofilm-related) infection.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the use of PCC0208009 in the preparation of drugs for treating Staphylococcus aureus and Enterococcus infections. Background Technology

[0002] Gram-positive pathogens are important pathogens causing clinical infections, with Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium being particularly common. These pathogens widely colonize the skin and mucous membranes of the human body. Once they invade internal tissues or the bloodstream, they can cause a variety of serious diseases, including skin and soft tissue infections, bacteremia, endocarditis, pneumonia, and urinary tract infections, making them one of the leading causes of community-acquired and hospital-acquired infections worldwide. However, with the widespread use of antibiotics, clinically isolated Gram-positive pathogens have developed resistance to most β-lactam antibiotics, with resistance to penicillin, methicillin, and vancomycin being the most common. More seriously, in recent years, there has been an increasing number of reports of strains resistant to "last-line" antibiotics such as linezolid and daptomycin. The emergence of multidrug-resistant strains significantly increases treatment costs and hospital stays, posing a significant challenge to clinical anti-infective therapy. Besides drug resistance, the biofilm-forming ability of Gram-positive pathogens (especially Staphylococcus aureus and Enterococcus) is another major challenge in clinical treatment. Bacterial biofilms are mainly composed of extracellular macromolecules such as lipids, proteins, and small amounts of carbohydrates, allowing bacteria to adhere to the surfaces of implanted medical devices such as artificial valves, pacemakers, and catheters, leading to chronic and persistent infections. Studies have shown that bacterial cells embedded in biofilms are far more resistant to antibiotics than planktonic cells, and conventional concentrations of antimicrobial drugs are often insufficient to effectively kill or eliminate them. Therefore, when searching for novel drugs to combat Gram-positive bacterial infections, it is essential to simultaneously consider the drug's inhibitory or bacteriostatic effects on bacterial biofilms.

[0003] Faced with the increasingly severe situation of drug resistance, the development of antibacterial drugs with novel mechanisms of action or new chemical skeletons is urgently needed. Structural optimization and activity extension of known active compounds are among the important strategies for discovering novel antibacterial candidates. Summary of the Invention

[0004] To address the above technical problems, this invention discloses the use of PCC0208009 in the preparation of drugs against Staphylococcus aureus and Enterococcus spp. PCC0208009 exhibits broad-spectrum growth inhibition against a variety of Gram-positive bacteria, including Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium, and can significantly inhibit the formation of Staphylococcus aureus biofilms. It has potential application value in the prevention and treatment of biofilm-related chronic infections and medical device-related infections.

[0005] The technical solution adopted by this invention is as follows:

[0006] The use of PCC0208009 in the preparation of drugs against Staphylococcus aureus and Enterococcus spp. infections, wherein PCC0208009, CAS number 1668565-74-9, and the Enterococcus spp. are at least one of Enterococcus faecalis and Enterococcus faecium. PCC0208009 exhibits excellent antibacterial activity against Gram-positive pathogenic bacteria such as Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium, with a minimum inhibitory concentration (MIC) of 6.25 μM to 12.5 μM. Furthermore, PCC0208009 can significantly inhibit the formation of Staphylococcus aureus biofilms.

[0007] The structural formula of PCC0208009 is shown in formula (1):

[0008] (1).

[0009] PCC0208009, Chemical Name: 1-[4-Diisobutylamino-2′-(1H-tetrazol-5-yl)-biphenyl-3-yl]-3-p-tolylurea, Molecular Formula: C 29 H 35 N7O, with a molecular weight of 497.63, belongs to the N-arylurea class of compounds. Its structural characteristics include a biphenyl skeleton, a tetrazolium ring, a diisobutylamino group, and a p-tolylurea group. Current research indicates that PCC0208009 is a highly effective inhibitor of indoleamine 2,3-dioxygenase (IDO), achieving a half-maximal inhibitory concentration (IC50) of [amount missing] against IDO in HeLa cells. 50The concentration of PCC0208009 is 4.52 nM, classifying it as an indirect IDO1 inhibitor. Furthermore, PCC0208009 has shown antitumor activity when used in combination with temozolomide, and its ability to alleviate neuropathic pain and its comorbidities by modulating synaptic plasticity in the anterior cingulate cortex and amygdala. However, to date, there are no reports of PCC0208009 inhibiting the growth and biofilm formation of Gram-positive pathogenic bacteria (including Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium), and its potential as an antibacterial drug, especially against drug-resistant Gram-positive bacterial infections, remains unexplored. In this invention, extensive experimental studies have revealed that PCC0208009 exhibits broad-spectrum growth inhibition against a variety of Gram-positive bacteria.

[0010] As a further improvement of the present invention, the concentration of PCC0208009 in the application system is not less than 3.125 μg / mL. Further, the concentration of PCC0208009 in the application system is not less than 6.25 μg / mL.

[0011] As a further improvement of the present invention, the drug is a pharmaceutical composition or preparation. Preferably, the dosage form of the drug includes injections, tablets, pills, capsules, suspensions, granules, sprays, or emulsions.

[0012] This invention also discloses the application of PCC0208009 in the preparation of coatings that inhibit Staphylococcus aureus and Enterococcus spp., the coatings being used on the surface of medical devices. The CAS number of PCC0208009 is 1668565-74-9, and its structural formula is shown in formula (1). PCC0208009 exhibits excellent antibacterial activity against Gram-positive pathogenic bacteria such as Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium, and can significantly inhibit the formation of Staphylococcus aureus biofilms.

[0013] As a further improvement of the present invention, the concentration of PCC0208009 in the coating is not less than 2 μg / mL. Further, the concentration of PCC0208009 in the coating is not less than 6.25 μg / mL.

[0014] This invention also discloses the application of PCC0208009 in the preparation of disinfectants that inhibit Staphylococcus aureus and Enterococcus spp. The CAS number of PCC0208009 is 1668565-74-9, and its structural formula is shown in formula (1). PCC0208009 exhibits excellent antibacterial activity against Gram-positive pathogenic bacteria such as Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium, and can significantly inhibit the formation of Staphylococcus aureus biofilm.

[0015] As a further improvement of the present invention, the concentration of PCC0208009 in the disinfectant is not less than 3.125 μg / mL. Further, the concentration of PCC0208009 in the disinfectant is not less than 6.25 μg / mL.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] This invention discloses a novel pharmaceutical use for compound PCC0208009. Specifically, PCC0208009 exhibits excellent antibacterial activity against Gram-positive pathogenic bacteria such as Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium, with a minimum inhibitory concentration (MIC) ranging from 6.25 μM to 12.5 μM. Furthermore, PCC0208009 significantly inhibits the formation of Staphylococcus aureus biofilms, suggesting its potential application value in the prevention and treatment of biofilm-related chronic infections and medical device-related infections. Regarding its antibacterial mechanism, PCC0208009 exhibits highly efficient bactericidal activity. Studies have shown that PCC0208009 exerts a rapid and significant bactericidal effect by altering the permeability of the Staphylococcus aureus cell membrane and disrupting the integrity of the bacterial cell wall / membrane. In addition, safety evaluation results show that PCC0208009 has good safety in normal mammalian cells. CCK-8 assays showed that PCC0208009 had a half-maximal inhibitory concentration (IC50) against human umbilical vein endothelial cells (HUVECs), human hepatic stellate cells (LX-2), and human embryonic kidney cells (293T). 50 The concentrations of the active ingredients (MICs) ranged from 70.13 μM to 115.0 μM, significantly higher than the MIC values, indicating that the compound has a wide therapeutic window and good drug-like safety. In conclusion, PCC0208009, as a compound with novel antibacterial activity, shows broad application prospects in the preparation of drugs for treating infections caused by Gram-positive bacteria such as Staphylococcus aureus, Enterococcus faecalis, and other Gram-positive bacteria, as well as biofilm-related infections. Attached Figure Description

[0018] Figure 1The figure shows the effect of PCC0208009 on the growth kinetics of Staphylococcus aureus in Example 2 of the present invention; wherein, (a) is the growth curve of methicillin-sensitive Staphylococcus aureus (MSSA) standard strain ATCC29213 after adding different concentrations of PCC0208009, (b) is the growth curve of methicillin-resistant Staphylococcus aureus (MRSA) USA300 after adding different concentrations of PCC0208009, (c) is the growth curve of Enterococcus faecalis standard strain ATCC29212 after adding different concentrations of PCC0208009, and (d) is the growth curve of vancomycin-resistant Enterococcus (VRE) strain V583 after adding different concentrations of PCC0208009.

[0019] Figure 2 The above are time-kill curves of PCC0208009 against Staphylococcus aureus and Enterococcus faecalis in Example 3 of the present invention. Among them, (a) is the 24-hour kill curve of PCC0208009 at different concentrations against MSSA standard strain ATCC29213, (b) is the 24-hour kill curve of PCC0208009 at different concentrations against MRSA USA300, (c) is the 24-hour kill curve of PCC0208009 at different concentrations against Enterococcus faecalis standard strain ATCC29212, and (d) is the 24-hour kill curve of PCC0208009 at different concentrations against VRE V583, with daptomycin as a positive control.

[0020] Figure 3 The figure shows the results of the inhibitory effect of PCC0208009 on the formation of Staphylococcus aureus biofilm in Example 4 of the present invention; wherein, (a) is the interference effect of different sub-inhibitory concentrations (1 / 8×MIC, 1 / 4×MIC, 1 / 2×MIC) of the drug on the formation of biofilm of 8 MSSA(a) bacteria; (b) is the interference effect of different sub-inhibitory concentrations (1 / 8×MIC, 1 / 4×MIC, 1 / 2×MIC) of the drug on the formation of biofilm of 8 MRSA bacteria.

[0021] Figure 4 The results of detecting the effect of PCC0208009 on the cell membrane permeability of Staphylococcus aureus USA300 using different methods in Example 5 of this invention are shown; (a) is the cell membrane potential obtained by propidium iodide (PI) staining method, and (b) is the cell membrane potential obtained by DiBAC4(3) method.

[0022] Figure 5This is a comparison of the microscopic morphology of Staphylococcus aureus USA300 and the control sample after 2 hours of treatment with PCC0208009 (4×MIC) in Example 5 of the present invention. (a) is a scanning electron microscope image, (b) is a transmission electron microscope image, the control is the blank sample without PCC0208009 (4×MIC), and PCC0208009 is the sample treated with PCC0208009 (4×MIC).

[0023] Figure 6 This is the result of the CCK-8 assay for evaluating the cytotoxicity of PCC0208009 in Example 6 of this invention; where (a), (b), and (c) are the results of the cytotoxicity evaluation of HUVEC, LX-2, and 293T cells, respectively, and (d), (e), and (f) are the half-maximal inhibitory concentrations (IC50) of the drug against the three normal mammalian cells in (a), (b), and (c), respectively. 50 (and cell survival rate curves). Detailed Implementation

[0024] The preferred embodiments of the present invention will be described in further detail below.

[0025] Example 1

[0026] The MIC values ​​of PCC0208009 against Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium were determined using the microbroth dilution method, following the procedures outlined in the Clinical and Laboratory Standards Institute (CLSI) Standards for Antimicrobial Susceptibility Testing by Dilution Method for Aerobic Bacteria. The test strains included clinical isolates of each of the aforementioned species, as well as standard reference strains used for quality control and result reliability verification: Staphylococcus aureus standard strain ATCC 29213 and Enterococcus faecalis standard strain ATCC 29212.

[0027] Experimental Procedure: Each clinical isolate and standard reference isolate was inoculated into fresh cationic MH broth (CAMHB) and cultured overnight at 37°C and 180 rpm with shaking. The overnight culture was collected and the turbidity was adjusted to 0.5 McFarland standard units (equivalent to approximately 1.0 × 10⁸ CFU / mL) using a turbidimetric tube or spectrophotometer. Subsequently, the bacterial culture was diluted 1:100 with CAMHB medium to the final inoculum concentration. 100 μL of the diluted bacterial culture was added to each well of a 96-well sterile microplate. PCC0208009 was dissolved in an appropriate amount of dimethyl sulfoxide (DMSO) and serially diluted with CAMHB medium to create 10 drug concentration gradients (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39 μM). 100 μL of the corresponding concentration was added to each well, bringing the final reaction volume per well to 200 μL. The final drug concentrations were the values ​​in the serial gradients. The following controls were also included: Positive control wells (growth control): 200 μL of CAMHB medium containing bacteria but without the test drug; Negative control wells (blank control): 200 μL of sterile CAMHB medium containing neither bacteria nor the drug; Solvent control wells: CAMHB medium containing bacteria and an equal volume of DMSO as the drug wells, to eliminate the influence of the solvent itself on bacterial growth. The MIC value of the standard reference strain against daptomycin was used as the quality control standard.

[0028] After inoculation, the 96-well plates were placed in a 37°C incubator and incubated for 18–20 hours. After incubation, bacterial sedimentation at the bottom of each well and changes in turbidity of the culture medium were observed visually. The minimum drug concentration required to completely inhibit the visible growth of the test strain and the standard reference strain was defined as the MIC value of PCC0208009 for that strain. All test strains and reference strains were independently measured at least three times, and consistent results were taken. If the MIC value of the standard reference strain against daptomycin was within the expected quality control range specified by CLSI, the experimental system was considered reliable, and the MIC determination results of the clinical isolates were valid.

[0029] Table 1. Minimum inhibitory concentration (μg / mL) of PCC0208009 against Gram-positive bacteria.

[0030]

[0031] Note: MRSA: methicillin-resistant Staphylococcus aureus; MSSA: methicillin-sensitive Staphylococcus aureus; E. faecalis: Enterococcus faecalis; E. faecium: Enterococcus faecium; n is the number of strains tested.

[0032] The MIC values ​​are shown in Table 1. It can be seen that PCC0208009 exhibits good antibacterial activity against various Gram-positive bacteria, including Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium. The MIC values ​​are mainly distributed between 6.25 μM and 12.5 μM, especially against MRSA. 50 and MIC 90 Both are 6.25μM.

[0033] Example 2

[0034] Experiment on the effect of PCC0208009 on the growth kinetics of Staphylococcus aureus and Enterococcus faecalis.

[0035] To systematically evaluate the inhibitory effect of PCC0208009 on the proliferation of Staphylococcus aureus and Enterococcus faecalis, this embodiment uses a fully automated growth curve analyzer to dynamically monitor the growth and development of bacteria at different drug concentrations.

[0036] The experimental procedure included: inoculating the test strains, including methicillin-sensitive Staphylococcus aureus standard strain ATCC29213, methicillin-resistant Staphylococcus aureus (MRSA) USA300, Enterococcus faecalis standard strain ATCC29212, and vancomycin-resistant Enterococcus VRE strain V583, into tryptone soybean broth (TSB) medium and incubating overnight at 37°C with shaking. The overnight culture was then diluted 1:1000 with fresh TSB medium and added to 96-well cell culture plates. Subsequently, PCC0208009 was added at specific concentrations of 1 / 8×MIC, 1 / 4×MIC, 1 / 2×MIC, and 1×MIC. The inoculated 96-well plates were placed in an automated growth curve analyzer and incubated at 37°C. The absorbance (OD) at 600 nm was automatically measured and recorded every hour. 600 The bacterial growth kinetics curve was plotted based on the density of planktonic bacteria in the culture medium. Drug-free bacterial culture was used as a negative control, and TSB medium without bacteria was used as a blank control.

[0037] Growth curve results as follows Figure 1 As shown, at a concentration of 1 / 2×MIC, PCC0208009 significantly slowed the growth rate of MSSA airborne bacteria and inhibited the final growth of ATCC29213, V583, and ATCC29212; when the drug concentration reached 1×MIC, the growth of Enterococcus faecalis and Staphylococcus aureus was completely inhibited. These results indicate that PCC0208009 has a clear inhibitory activity against Staphylococcus aureus and Enterococcus faecalis, and exhibits a more significant growth-inhibiting effect on drug-resistant strains, suggesting its potential as a therapeutic agent against Gram-positive bacterial infections (especially drug-resistant strains).

[0038] Example 3

[0039] Phytokinetic analysis of PCC0208009 against Staphylococcus aureus and Enterococcus faecalis.

[0040] To evaluate the bactericidal activity and kinetic characteristics of PCC0208009 against Gram-positive pathogens, this embodiment uses the time-kill assay, with daptomycin, a commonly used clinical antibacterial drug, as a positive control, to systematically investigate the bactericidal effect of PCC0208009 against Staphylococcus aureus (including methicillin-sensitive and methicillin-resistant strains) and Enterococcus faecalis.

[0041] The experimental procedure includes: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 600 Test strains with a concentration ≈ 0.5 (including methicillin-sensitive Staphylococcus aureus (MSSA) CHS101, methicillin-resistant Staphylococcus aureus (MRSA) USA300, and the standard strain of Enterococcus faecalis ATCC29212, and the vancomycin-resistant Enterococcus VRE strain V583) were diluted 100-fold with fresh TSB medium. They were then incubated in the following groups: the diluted bacterial solutions were mixed with serial concentrations of PCC0208009 (2×MIC, 4×MIC) and daptomycin (4×MIC), and incubated at 37°C and 200 rpm with shaking. Bacterial samples were collected at 0, 2, 6, and 24 hours of incubation. After serial 10-fold dilutions with sterile physiological saline, appropriate amounts of the diluted solutions were spread onto TSB agar plates and incubated at 37°C for 24 hours. Colony counting was performed, and results were expressed as CFU / mL. Biological replicates were established for each time point and each drug concentration.

[0042] The sterilization curve results are as follows Figure 2 As shown, PCC0208009 exhibited clear bactericidal activity against the tested strains at a concentration of 2×MIC; against ATCC29212 and ATCC29213, it exerted significant bactericidal activity at a concentration of 4×MIC, reducing viable bacterial counts to the detection limit (<10 CFU / mL) within 24 hours of incubation, demonstrating superior bactericidal efficacy compared to daptomycin at 4×MIC. Furthermore, PCC0208009 also showed significant bactericidal activity against MRSA-resistant strain USA300 and VRE-resistant strain V583. These results indicate that PCC0208009 possesses rapid and efficient bactericidal activity against Staphylococcus aureus (including resistant strains) and Enterococcus faecalis, exhibiting superior bactericidal kinetics against resistant strains, suggesting its potential as a candidate drug for treating infections caused by Gram-positive bacteria (especially resistant strains).

[0043] Example 4

[0044] Experiment on the effect of PCC0208009 on biofilm formation of Staphylococcus aureus.

[0045] Bacterial biofilm formation is a major cause of clinical anti-infective therapy failure and persistent chronic infections. Therefore, when evaluating the drug-likeness of novel antimicrobial compounds, it is essential to systematically examine their ability to inhibit bacterial biofilm formation or maturation.

[0046] This embodiment uses the classic crystal violet staining method to quantitatively evaluate the effect of sub-inhibitory concentrations of PCC0208009 on biofilm formation in Staphylococcus aureus. The experimental steps included: Overnight cultured Staphylococcus aureus was diluted 1000-fold with TSB medium (TSBG) containing 2% glucose. The diluted bacterial solution was added to 96-well cell culture plates, 200 μL per well. A series of sub-inhibitory concentrations of PCC0208009 (1 / 8×MIC, 1 / 4×MIC, 1 / 2×MIC, etc.) were added to the experimental groups, with three replicates per group. The following controls were also set up: negative control (blank control was TSBG medium without bacterial culture); solvent control (growth control) was TSBG medium containing bacterial culture and an equal volume of DMSO solvent as the drug group. The inoculated 96-well plates were placed in a 37°C incubator for 24 hours. After incubation, the culture medium in the wells was gently aspirated, and the plates were gently washed three times with PBS to remove unadhered airborne bacteria, then air-dried at room temperature. Methanol was added to each well for fixation for 15 minutes, the methanol was aspirated, and then 1% (w / v) crystal violet staining solution was added for staining for 15 minutes. After staining, the plates were rinsed three times with sterile distilled water until the eluent from the negative control wells was colorless, and then air-dried at room temperature. Finally, 200 μL of anhydrous ethanol was added to each well, and the plates were shaken at room temperature for 1 minute to fully dissolve the crystal violet. The absorbance (OD) at 570 nm was measured using a microplate reader. 570 The experiment was independently repeated 3 times, with 3 replicates each time, and the average value was taken as the final quantitative result of biofilm.

[0047] The results are as follows Figure 3 As shown in the figure, within the sub-inhibitory concentration range, PCC0208009 inhibited the formation of Staphylococcus aureus biofilms in a concentration-dependent manner. When the concentration of PCC0208009 reached 1 / 2 × MIC, it inhibited the formation of Staphylococcus aureus biofilms in all tested strains (including MRSA and MSSA). These results indicate that PCC0208009 can specifically interfere with or inhibit bacterial biofilm formation at concentrations below those inhibiting planktonic bacterial growth, suggesting its unique application potential in the prevention and treatment of Staphylococcus aureus biofilm-related chronic infections and medical device-related infections.

[0048] Example 5

[0049] Experiment on the effect of PCC0208009 on the permeability and membrane potential of Staphylococcus aureus cell membrane.

[0050] To investigate the antibacterial mechanism of PCC0208009, this embodiment uses propidium iodide (PI) permeation experiment, DiBAC4(3) membrane potential detection experiment, and scanning and transmission electron microscopy to systematically evaluate the effects of PCC0208009 on the cell membrane structure and function of Staphylococcus aureus.

[0051] 1. Cell membrane permeability assay (PI staining method)

[0052] Propidium iodide (PI) is an ethidium bromide analog and a commonly used nuclear staining reagent. PI itself cannot penetrate living cells with intact membrane structures, but when cell membrane integrity is impaired and permeability increases, PI can enter the cell and bind to double-stranded DNA, releasing red fluorescence. Therefore, an increase in PI fluorescence intensity can reflect changes in cell membrane permeability and is often used to differentiate between early apoptotic cells and late apoptotic / necrotic cells (late apoptotic and necrotic cells have significantly increased membrane permeability).

[0053] Experimental method: Staphylococcus aureus USA300 cell suspension in logarithmic growth phase was adjusted to OD. 600 =0.05, and incubated with PI solution. Subsequently, different concentrations of PCC0208009 (final concentrations of 1×MIC and 4×MIC) were added to each group. A 0.1% DMSO solvent control and a 0.1% Triton X-100 (cell membrane disruptor) positive control were also included. Fluorescence intensity changes in each group were continuously monitored using a fluorescence microplate reader, with the excitation wavelength set at 535 nm and the emission wavelength at 617 nm.

[0054] The results are as follows Figure 4 As shown in Figure a, after treatment with PCC0208009, the PI fluorescence value showed a gradient increasing trend with the increase of drug concentration, suggesting that PCC0208009 can change the permeability of Staphylococcus aureus cell membrane to a certain extent.

[0055] 2. Measurement of cell membrane potential (DiBAC4(3) method)

[0056] DiBAC4(3) is a lipophilic anionic fluorescent dye used to detect cell membrane potential. The dye itself is non-fluorescent, but it emits fluorescence after entering the cell and binding to proteins in the cytoplasm. Increased intracellular fluorescence intensity indicates an elevated membrane potential (depolarization); conversely, decreased fluorescence intensity indicates a decreased membrane potential (hyperpolarization).

[0057] The experimental procedure included adjusting the suspension of Staphylococcus aureus USA300 cells in the logarithmic growth phase to OD. 600 = 0.05, add DiBAC4(3) to a final concentration of 2 μM, and incubate at room temperature for 10 minutes. Then add different concentrations of PCC0208009 (final concentrations of 1×MIC and 4×MIC). A 0.1% DMSO solvent control and a 0.1% Triton X-100 control were also set up. Fluorescence intensity changes were monitored using a fluorescence microplate reader at an excitation wavelength of 490 nm and an emission wavelength of 515 nm.

[0058] The results are as follows Figure 4 As shown in b. After treatment with PCC0208009, the fluorescence intensity of DiBAC4(3) of MRSA strain USA300 gradually increased with the increase of drug concentration, showing a gradient depolarization trend.

[0059] 3. Bacterial morphological observation (scanning electron microscopy and transmission electron microscopy)

[0060] To further and visually verify the destructive effect of PCC0208009 on the cell membrane structure of Staphylococcus aureus, this experiment used scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to observe the changes in the ultrastructure of bacteria before and after drug treatment.

[0061] Experimental Methods: Staphylococcus aureus USA300 cell suspension in logarithmic growth phase was adjusted to a suitable concentration, and PCC0208009 was added to a final concentration of 4×MIC. The cells were then treated with shaking at 37℃ and 200 rpm for 2 hours. A negative control group (containing only an equal volume of DMSO solvent) was also included. After treatment, bacterial cells from each group were collected, washed with PBS, and then fixed, dehydrated, dried, and embedded for sectioning according to standard sample preparation procedures for scanning electron microscopy and transmission electron microscopy, respectively.

[0062] The results are as follows Figure 5 As shown in Figure a. Scanning electron microscopy revealed that the untreated negative control group bacteria were typically spherical or ellipsoidal in shape, with smooth, plump surfaces and intact morphology. However, after treatment with 4×MIC PCC0208009 for 2 hours, a large number of bacterial cells showed significant morphological changes, with wrinkled and sunken cell surfaces, and numerous broken cells and cell debris, indicating bacterial cell membrane rupture. Transmission electron microscopy. Figure 5 b further showed that the bacterial cell walls and cell membranes of the control group were intact and the cytoplasm was evenly distributed; while the drug-treated group showed severe damage to the cell membrane structure, separation or rupture of the cell wall from the cell membrane, leakage of cytoplasmic contents, and complete disintegration of some cells.

[0063] The experimental data from this embodiment demonstrate that PCC0208009 exerts significant antibacterial and bactericidal effects by altering the permeability of the Staphylococcus aureus cell membrane, inducing cell membrane hyperpolarization, and directly disrupting the morphological integrity of the bacterial cell membrane, leading to leakage of cell contents and bacterial death. These results, from both functional and morphological perspectives, confirm the antibacterial mechanism of PCC0208009, which targets the bacterial cell membrane as a key site.

[0064] Example 6

[0065] Evaluation of the toxicity of PCC0208009 to mammalian cells.

[0066] To evaluate the safety and therapeutic window of PCC0208009 as an antibacterial drug, this embodiment used the CCK-8 assay to detect the cytotoxicity of PCC0208009 on human umbilical vein endothelial cells (HUVEC), human hepatic stellate cells (LX-2), and human renal epithelial cell line (293T).

[0067] The experimental procedure included: HUVEC, LX-2, and 293T cells in logarithmic growth phase were seeded at appropriate densities into 96-well cell culture plates, 100 μL per well, and pre-cultured at 37°C in a 5% CO2 incubator for 24 hours to allow cell adhesion. Subsequently, 10 μL of PCC0208009 solution at serial concentrations was added to each well, resulting in final concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.13, and 1.56 μM, with three replicates for each concentration. The culture plates were then incubated at 37°C in a 5% CO2 incubator for another 24 hours. After incubation, 10 μL of CCK-8 working solution was added to each well, and incubation continued for 1–4 hours. The absorbance (OD450) of each well was measured using a microplate reader. The CCK-8 kit is based on the WST-8 colorimetric reaction. In the presence of an electron coupling reagent, WST-8 can be reduced by dehydrogenases in the mitochondria of living cells to produce a highly water-soluble, orange-yellow formazan product. The amount of formazan produced is positively correlated with the number of living cells and cellular metabolic activity, and negatively correlated with cytotoxicity. Cell viability can be indirectly reflected by measuring the absorbance at 450 nm.

[0068] The results are as follows Figure 6 As shown in ac, 25.0 μM PCC0208009 showed no significant toxicity to the proliferation of the three cell types tested. The results were calculated using a fitting method. Figure 6 df, the half-maximal inhibitory concentration (IC50) of PCC0208009 in HUVEC, LX-2 and 293T cells 50 The corresponding values ​​are 104.8 μM, 115.0 μM, and 70.13 μM. The above IC... 50The values ​​were all significantly higher than the MIC values ​​of PCC0208009 against the tested Gram-positive pathogens, indicating that the compound has good safety against normal mammalian cells at effective antibacterial concentrations, and possesses a wide therapeutic window and potential clinical anti-infective application value.

[0069] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. The use of PCC0208009 in the preparation of drugs for treating Staphylococcus aureus and Enterococcus spp. infections, characterized in that: The PCC0208009 has a CAS number of 1668565-74-9; the Enterococcus genus is at least one of Enterococcus faecalis and Enterococcus faecium.

2. The use of PCC0208009 according to claim 1 in the preparation of drugs for treating Staphylococcus aureus and Enterococcus infections, characterized in that: The concentration of PCC0208009 in the application system is not less than 3.125 μg / mL.

3. The use of PCC0208009 according to claim 2 in the preparation of drugs for treating Staphylococcus aureus and Enterococcus infections, characterized in that: The drug is an injection, tablet, pill, capsule, suspension, granule, spray, or emulsion.

4. The application of PCC0208009 in the preparation of coatings that inhibit Staphylococcus aureus and Enterococcus spp., characterized in that: The coating is used on the surface of medical devices. The CAS number of PCC0208009 is 1668565-74-9. The Enterococcus species is at least one of Enterococcus faecalis and Enterococcus faecium.

5. The application of PCC0208009 according to claim 4 in the preparation of coatings that inhibit Staphylococcus aureus and Enterococcus, characterized in that: In the coating, the concentration of PCC0208009 is not less than 3.125 μg / mL.

6. PCC0208009 is used in the preparation of disinfectants that inhibit Staphylococcus aureus and Enterococcus spp., characterized in that: The CAS number of PCC0208009 is 1668565-74-9, and the Enterococcus genus is at least one of Enterococcus faecalis and Enterococcus faecium.

7. The application of PCC0208009 according to claim 6 in the preparation of disinfectants that inhibit Staphylococcus aureus and Enterococcus spp., characterized in that: In the disinfectant, the concentration of PCC0208009 is not less than 3.125 μg / mL.

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