Use of sc-43 for the preparation of a medicament for the treatment of gram-positive bacterial infections

Abstract

The application provides application of SC-43 in preparation of an anti-Gram-positive bacterial infection drug, wherein the SC-43 has a CAS number of 1400989-25-4; the SC-43 has the effects of inhibiting growth and biofilm formation of Gram-positive bacteria. The technical scheme of the application discloses a new medical use of SC-43; the SC-43 has better antibacterial activity on various Gram-positive bacteria and can significantly inhibit biofilm formation; the SC-43 has antibacterial activity comparable to Van (vancomycin) and better bactericidal activity than Van, and has high biological activity of killing Staphylococcus aureus; the SC-43 can affect membrane permeability.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the application of SC-43 in the preparation of drugs for treating Gram-positive bacterial infections. Background Technology

[0002] Gram-positive bacteria are among the most common pathogens, and the infectious diseases they cause pose a significant threat to humans. They can cause a range of diseases, including local soft tissue purulent infections, pneumonia, endocarditis, osteomyelitis, and septic arthritis. In severe cases, they can lead to bacteremia, septic shock, and even death. According to the latest 2021 National Antimicrobial Resistance Surveillance Network (CARSS) report, Staphylococcus aureus (S. aureus) ranked first in isolation rate among Gram-positive bacteria, accounting for 32.6% of all Gram-positive bacteria. This was followed by Enterococcus faecium (E. faecium), Enterococcus faecalis (E. faecalis), Staphylococcus epidermidis (S. epidermidis), and Streptococcus pneumoniae (S. pneumoniae). The national average detection rate of methicillin-resistant Staphylococcus aureus (MRSA) was 29.4%. The latest CHINET China Antimicrobial Resistance Surveillance results released in 2023 indicate that among the 339,513 clinically isolated bacteria, the Gram-positive isolates, ranked by the number of isolates, were: Staphylococcus aureus (9.47%), Enterococcus faecalis (4.31%), Enterococcus faecium (3.60%), Streptococcus pneumoniae (2.64%), Staphylococcus epidermidis (2.06%), Streptococcus agalactiae (1.79%), and Staphylococcus hominis (1.19%). The detection rate of methicillin-resistant Staphylococcus aureus (MRSA) remained at 28.7%, indicating that drug resistance remains a serious and widespread problem, and is higher than that of methicillin-susceptible Staphylococcus aureus (MSSA). The detection rate of *Staphylococus epidermidis* (MRSE) remains as high as 82.2%; strains of *Enterococcus faecalis* and *Enterococcus faecium* have emerged that are resistant to almost all clinically used antibiotics, and the resistance rate of *Enterococcus faecalis* to vancomycin (Van) has increased from 1.4% in 2021 to 2.2%, while that of *Enterococcus faecalis* is 0.1%. The situation of bacterial resistance to antimicrobial drugs is becoming increasingly serious and widespread. Infections caused by drug-resistant Gram-positive bacteria remain a huge burden on the public health system, and antibiotic resistance has become one of the important reasons for poor treatment outcomes or treatment failures.

[0003] To address the looming global drug resistance crisis, the development of novel antibacterial drugs that effectively inhibit Gram-positive bacteria has become an urgent need. Furthermore, the formation of bacterial biofilms (BF) and persistent bacteria are both challenging issues contributing to bacterial resistance and impacting clinical treatment. Therefore, developing novel antibacterial drugs that effectively inhibit drug-resistant bacteria, kill persistent bacteria, and inhibit or even eliminate biofilm formation, as well as exploring optimal targeted drug delivery methods, has become a current research hotspot and a serious challenge and urgent task facing clinical practice. Summary of the Invention

[0004] To address the above technical problems, this invention discloses the application of SC-43 in the preparation of drugs against Gram-positive bacterial infections. SC-43 has highly efficient activity against the growth of Gram-positive bacteria and against biofilm formation.

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

[0006] SC-43 is used in the preparation of drugs against Gram-positive bacterial infections. SC-43 has the CAS number 1400989-25-4. SC-43 has the function of inhibiting the growth of Gram-positive bacteria and biofilm formation.

[0007] The structural formula of the SC-43 is shown in formula (1):

[0008]

[0009] SC-43 is a novel, multi-targeted oral anti-tumor derivative of sorafenib, a potent orally active SHP-1 (Src-homology protein tyrosine phosphatase-1) agonist. Studies have shown that SC-43 significantly enhances SHP-1 activity and downregulates phosphorylated STAT3 levels, demonstrating effectiveness in inhibiting the in vitro stemness of human colorectal cancer cells, making it a potentially highly effective treatment for this disease. SC-43 exhibits higher activity against advanced hepatocellular carcinoma than sorafenib, inducing significant apoptosis in sorafenib-resistant cells and showing better survival benefits than sorafenib in orthotopic advanced hepatocellular carcinoma. Developing a novel combination therapy by conjugating sorafenib with SC-43 resulted in greater reduction in tumor size and prolonged survival in mice with orthotopic advanced hepatocellular carcinoma. Combining SC-43 with docetaxel also showed enhanced tumor growth inhibition. Furthermore, SC-43 can induce apoptosis in cholangiocarcinoma cells through the SHP-1 / STAT3 signaling pathway. This suggests that SC-43 plays an important role in anti-tumor activity. However, there are currently no reports on the antibacterial effects of SC-43.

[0010] As a further improvement of the present invention, the Gram-positive bacteria are at least one of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Streptococcus agalactiae, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus lysus, and Staphylococcus capitella. Numerous experiments have confirmed that SC-43 can effectively inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA), Enterococcus faecalis, Enterococcus faecium, Streptococcus agalactiae, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus lysus, and Staphylococcus capitella in vitro, and also inhibit biofilm formation, demonstrating highly effective biological activity in killing Staphylococcus aureus. Regarding the mechanism, multiple experiments have demonstrated that it may target the cell membrane.

[0011] As a further improvement of the present invention, the concentration of SC-43 in the treatment system is not less than 0.39 μM.

[0012] As a further improvement of the present invention, the drug is a pharmaceutical composition or formulation. Further, the drug is an injection, tablet, pill, capsule, suspension, granule, spray, or emulsion.

[0013] The present invention also discloses the application of SC-43 in the preparation of coatings that inhibit Gram-positive bacteria, the coatings being used on the surface of medical devices, the CAS number of SC-43 being 1400989-25-4, and the structural formula being shown in formula (1); the SC-43 has the function of inhibiting the growth of Gram-positive bacteria and the formation of biofilms.

[0014] As a further improvement of the present invention, the concentration of SC-43 in the coating is not less than 0.39 μM.

[0015] The present invention also discloses the application of SC-43 in the preparation of antibacterial agents against Gram-positive bacteria. The CAS number of SC-43 is 1400989-25-4, and its structural formula is shown in formula (1). SC-43 has the function of inhibiting the growth of Gram-positive bacteria and the formation of biofilms.

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

[0017] The present invention discloses a novel pharmaceutical use for SC-43. SC-43 exhibits good antibacterial and anti-biofilm activity against a variety of Gram-positive bacteria. SC-43 has antibacterial activity comparable to Vancomycin and superior bactericidal activity, inhibiting biofilm formation and demonstrating highly efficient biological activity in killing Staphylococcus aureus. SC-43 can affect membrane permeability, making it a potential candidate as a highly effective and broad-spectrum novel antibacterial drug.

[0018] Further investigation into the SC-43 mechanism revealed that SC-43 can induce bacterial membrane rupture and alter membrane permeability. The MIC of SC-43 against bacteria significantly increased in a concentration-dependent manner after the addition of different membrane phospholipids. Finally, proteomic analysis identified 40 upregulated proteins and 54 downregulated proteins. Among the 40 upregulated proteins, 3 were transmembrane proteins (one of which was predicted), 2 were subcellularly localized on the membrane, and 1 was associated with the cell wall. Therefore, 4 of the upregulated proteins were associated with the membrane and cell wall. Of the 54 downregulated proteins, 14 were transmembrane proteins, 15 were subcellularly localized on the membrane (9 of which were both transmembrane and membrane-associated), and 1 was located on the cell wall. Therefore, 20 of the downregulated proteins were associated with the membrane and cell wall. In summary, a total of 25.5% of the differentially expressed proteins were associated with the membrane and cell wall. Cellular component analysis mainly focused on the membrane, periplasmic space of the outer membrane, and some transport complexes; KEGG analysis showed only one One Carbon Pool by folate pathway, and biological process analysis showed that it was related to tetrahydrofolate conversion and single-carbon metabolism, thus further proving that it may target the membrane to exert antibacterial effects. Attached Figure Description

[0019] Figure 1 This paper presents the effects of different concentrations of SC-43 on the growth curves of Staphylococcus aureus MRSA and MSSA in embodiments of the present invention; where A, B, C, and D are the growth curves of strains CHS684, CHS791, YUSA213, and YUSA128, respectively; data are expressed as mean ± SEM. N = 3.

[0020] Figure 2 This is a graph showing the experimental results of the biofilm activity analysis of SC-43 against Staphylococcus aureus in this invention. In the graph, A represents the OD595 value of MSSA strains after treatment with SC-43; B represents the OD570 value of MSSA strains after treatment with SC-43; C represents the OD595 value of MRSA strains after treatment with SC-43; and D represents the OD570 value of MRSA strains after treatment with SC-43. Except for strain CHS791 (N=13), all other strains have N=6. Data are expressed as Mean ± SEM. *P<0.05, **P<0.01 # P<0.0001.

[0021] Figure 3This is a diagram illustrating the bactericidal effect of SC-43 against Staphylococcus aureus according to an embodiment of the present invention; wherein, A is the bactericidal curve of SC-43 treating the logarithmic-phase SA113 strain; B is the bactericidal curve of SC-43 treating the logarithmic-phase YUSA145 strain; C is the bactericidal curve of SC-43 treating the plateau-phase SA113 strain; and D is the bactericidal curve of SC-43 treating the plateau-phase YUSA145 strain. All data are expressed as Mean+SEM. N = 2-3.

[0022] Figure 4 These are the drug safety test results of SC-43 according to embodiments of the present invention; wherein, A represents the toxicity result of SC-43 on A549 cells; B represents the toxicity result of SC-43 on HepG2 cells; C represents the toxicity result of SC-43 on LX-2 cells; D represents the toxicity result of SC-43 on J774A.1 cells; E represents the toxicity result of SC-43 on 293T cells; F represents the toxicity result of SC-43 on BEAS-2B cells. N=6. All values ​​are Mean±SEM; G is the hemolysis status graph; H is the hemolysis rate. N=4. All values ​​are Mean±SEM.

[0023] Figure 5 These are the results of SC-43 targeting cell membranes in this embodiment of the invention; wherein, A is the permeability of SA113 membrane after SC-43 treatment; B is the permeability of YUSA145 membrane after SC-43 treatment; C is the fold change of SC-43 on the minimum inhibitory concentration (MIC) of SA113 after the addition of different concentrations of membrane phospholipids; and D is the fold change of SC-43 on the MIC after the addition of different concentrations of membrane phospholipids.

[0024] Figure 6 These are electron micrographs of the changes in the membrane of the SC-43 treated strain 2 hours after the present invention. Among them, A is the effect of SC-43 on the membrane of the YUSA145 strain; B is the effect of SC-43 on the membrane of the EF16C51 strain.

[0025] Figure 7 This is the result of differential protein analysis of Staphylococcus aureus YUSA145 after treatment with SC-43 according to an embodiment of the present invention; wherein, A is a volcano plot of differential proteins; B is a bar chart of the number of differential proteins; and C is a pathway diagram of biological process enrichment analysis. The cutoff value is set to 1.2-fold differential expression.

[0026] Figure 8This is another differential protein analysis result after treating Staphylococcus aureus YUSA145 with SC-43 according to an embodiment of the present invention; wherein, A is the KEGG enrichment analysis pathway diagram; B is the molecular function enrichment analysis pathway diagram; C is the cellular component enrichment analysis pathway diagram; D is the gene proportion of the cellular component enrichment analysis pathway diagram; E is the protein-protein interaction (PPI) analysis. The cutoff value is set to 1.2-fold differential expression. Detailed Implementation

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

[0028] Example 1

[0029] According to the performance standards for antimicrobial susceptibility testing in the 31st edition of the Clinical and Laboratory Standards Institute (CLSI) guidelines, CAMHB medium was used, and the MICs of SC-43 against 13 Staphylococcus aureus MRSA strains, 9 MSSA strains, 19 Enterococcus faecalis strains, and 14 Staphylococcus epidermidis strains were determined using the microbroth dilution method, with vancomycin as a control. Specific steps included:

[0030] The minimum inhibitory concentration (MIC) of SC-43 against experimental strains was determined according to the performance standards for antimicrobial susceptibility testing in the 31st edition of the Clinical and Laboratory Standards Institute (CLSI) guidelines. The MIC against clinical isolates was determined using CAMHB medium and the broth dilution method. A specially designed honeycomb 100-well plate was used, with the concentration array configured as follows: 1×MIC, 1 / 2×MIC, 1 / 4×MIC, 1 / 8×MIC, control, and Blank (each...). (3 biological replicates); Drug dilution: Add 200 μl of the prepared drug dilution to each of the first rows, and take 100 μl from the first row to the corresponding concentration, and mix by pipetting 10 times each; Add 100 μl of TSB bacterial solution diluted 1:1000 to the sample group, control group and Blank group respectively; Final concentration 1:2000; Place in a fully automated microbial growth analyzer (Finland), select all wells, incubate at 37℃ for 24 h, and compare the growth of planktonic bacteria under different conditions by measuring the absorbance at 600 nm wavelength per hour.

[0031] The MIC values ​​are shown in Table 1, and the distribution of the minimum inhibitory concentrations (MICs) of SC-43 and van against Staphylococcus aureus strains is shown in Table 2. It is evident that SC-43 exhibits very high inhibitory activity against almost all Gram-positive bacteria. The MIC range for SC-43 against 13 Staphylococcus aureus MRSA strains, 9 MSSA strains, 19 Enterococcus faecalis strains, and 14 Staphylococcus epidermidis strains is 0.39-1.56 μM. In contrast, the corresponding first-line clinical antibiotic, vancomycin (Van), has an MIC range of 1-4 μg / ml (0.69-2.76 μM) against the same strains, showing slightly lower efficacy than SC-43. For 13 Streptococcus agalactiae strains, the MIC ranges for SC-43 and Van are 0.39-0.78 μM and 0.25-0.5 μg / ml, respectively, with Van showing slightly better efficacy than SC-43. For 15 strains of Staphylococcus aureus, the MIC ranges of SC-43 and Van were 0.39–1.56 μM and 0.5–2 μg / ml (0.345–1.38 μM), respectively, showing similar efficacy. For 4 strains of Staphylococcus capitulata, the MIC ranges of SC-43 (0.39–0.78 μM) and Van (0.5–1 μg / ml, i.e., 0.345–0.69 μM) were similar. For 15 strains of Enterococcus faecalis, the MIC ranges of SC-43 and Van were 0.78–1.56 μM and 0.5–1 μg / ml (0.345–0.69 μM), respectively. For 4 strains of Staphylococcus hemolyticus, the MIC range of SC-43 was 0.78–3.125 μM, and the MIC range of Van was 1–2 μg / ml (0.69–1.38 μM).

[0032] Table 1. Minimum inhibitory concentrations (MICs) of SC-43 against Staphylococcus aureus, Streptococcus agalactiae, Enterococcus faecalis, Enterococcus gasseri, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus capitella, and Staphylococcus hemolyticus strains.

[0033]

[0034] Table 2. Distribution of minimum inhibitory concentrations (MICs) of SC-43 against Staphylococcus aureus strains

[0035]

[0036] Note: MRSA: methicillin-resistant Staphylococcus aureus; MSSA: methicillin-sensitive Staphylococcus aureus.

[0037] In summary, SC-43 exhibits high inhibitory activity against the aforementioned Gram-positive bacteria. Its MIC50 against Staphylococcus aureus (MRSA), MSSA, Streptococcus agalactiae, Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus capitella is 0.78 μM, while its MIC50 against hemolytic Staphylococcus is 1.56 μM. In contrast, the clinical-grade antibiotic vancomycin has an MIC50 of 1 μg / ml (0.69 μM) against MRSA, MSSA, Enterococcus faecalis, Enterococcus faecium, Staphylococcus hominis, and Staphylococcus capitella, except against Streptococcus agalactiae (MIC50 = 0.25 μg / ml) and Staphylococcus epidermidis (MIC50 = 2 μg / ml, i.e., 1.38 μM). Therefore, by comparing the MICs of numerous Gram-positive bacterial strains, it can be concluded that the antibacterial activity of SC-43 is almost comparable to that of the first-line clinical antibiotic vancomycin, exhibiting very high inhibitory activity against Gram-positive bacteria and possessing significant research value.

[0038] In addition, the effective group of SC-43 may be phenoxy, so some drugs containing phenoxy structures were selected to detect its minimum inhibitory concentration (MIC), as shown in Table 3.

[0039] The results showed that while the analogues Triclabendazole and TAK-285 exhibited some antibacterial activity against Staphylococcus aureus and Enterococcus faecalis, their effectiveness was far lower than that of SC-43. Several other analogues containing phenoxy groups showed almost no antibacterial activity.

[0040] Table 3. Minimum MIC of SC-43 analogs for some representative bacteria

[0041]

[0042] Example 2

[0043] Experiment on the effect of SC-43 on the growth of Staphylococcus aureus MRSA and MSSA.

[0044] To verify whether SC-43 can inhibit the growth of Staphylococcus aureus, this experiment treated Staphylococcus aureus MRSA strains CHS684, CHS791, YUSA213 and MSSA strain YUSA128 with different concentrations (1 / 8×MIC-1×MIC) of SC-43, and measured their OD values ​​every two hours within 0-24h.

[0045] Specific steps: Dilute the overnight cultured bacterial solution at a ratio of 1:1000 and add it to a specially made honeycomb 100-well plate (with 3 replicates). Then, dilute SC-43 drug to a certain concentration (1×MIC, 1 / 2×MIC, 1 / 4×MIC, 1 / 8×MIC, 1 / 16×MIC) and add it to the well plate in equal volumes. Place the well plate in a growth curve analyzer and continuously measure the OD600 absorbance for 24 hours. Plot the growth curve of each strain based on the measured values.

[0046] The obtained growth curve is as follows Figure 1 As shown, SC-43 completely inhibited the growth of CHS684 (1.56 μM), CHS791 (0.78 μM), YUSA213 (0.78 μM), and YUSA128 (0.78 μM) at 1×MIC. At a sub-inhibitory concentration of 1 / 2×MIC, SC-43 (0.78 μM) completely inhibited the growth of CHS684. However, for the YUSA213 strain, 1 / 2×MIC (0.39 μM) completely inhibited the growth of the strain in the first 20 hours, but the bacterial count rebounded after 20-24 hours, failing to achieve complete inhibition. For the CHS791 and YUSA128 strains, 1 / 2×MIC (0.39 μM) had almost no inhibitory effect on the growth of the strains. It significantly inhibited the growth of YUSA145. For YUSA145 (0.78 μM), it completely inhibited the growth in the early stages, but a slight bacterial growth was observed after 22-24 hours. The results showed that SC-43 could effectively inhibit the growth of Staphylococcus aureus MRSA and MSSA.

[0047] Example 3

[0048] Experiment on the effect of SC-43 on Staphylococcus aureus biofilm formation.

[0049] Biofilm formation is a key challenge in anti-infective therapy; therefore, assessing drug effectiveness requires monitoring its ability to inhibit biofilm formation. In this example, Staphylococcus aureus MSSA and MRSA strains were treated with different concentrations of SC-43 for 24 hours. OD595 was measured to detect bacterial growth. After fixation, crystal violet staining was used, and after drying, OD570 was measured to detect biofilm formation. Specific steps included:

[0050] Five MSSA and five MRSA strains cultured overnight were diluted 1:100 with TSBG and added to 96-well plates. 100 μl of SC-43 diluted at a certain factor was then added, with a blank control group included. After static incubation at 37℃ for 24 h, the OD595 values ​​of bacteria treated with different concentrations of SC-43 were measured to assess bacterial growth. The supernatant was removed, and the plates were washed three times with 100 μl of sterile water to remove airborne bacteria. The plates were dried and fixed with 100 μl of methanol for 15 min. After slightly drying, 100 μl of 1% crystal violet was added for staining for 15 min. The crystal violet was washed away, and the plates were dried again. The OD570 values ​​of the biofilms treated with different concentrations of SC-43 were measured to assess bacterial biofilm formation.

[0051] The results are as follows Figure 2 As shown, SC-43 can reduce biofilm formation at a sub-inhibitory concentration that does not inhibit bacterial growth. Figure 2 In group A, MSSA strains were treated with SC-43 at sub-inhibitory concentrations of 1 / 16×MIC, 1 / 8×MIC, 1 / 4×MIC, and 1 / 2×MIC. It was found that the OD595 value did not decrease, indicating that the bacterial load did not decrease. Figure 2 As shown in Figure B, biofilm formation was significantly reduced. 1 / 16×MIC significantly reduced the biofilm of YUSA135, 1 / 8×MIC significantly reduced the biofilm of YUSA128 and YUSA135, and 1 / 8×MIC significantly reduced the biofilm of SA113, YUSA128, YUSA135, and YUSA13. SC-43 at 1 / 4×MIC and 1 / 2×MIC significantly reduced the biofilm of the five MSSA strains shown in the figure. At a concentration of 1×MIC, SC-43 greatly reduced the bacterial cell count, thus leading to a reduction in biofilm. Figure 2 As shown in C, for MRSA strains, the subinhibitory concentration of SC-43 does not change the bacterial count, but it can significantly inhibit biofilm formation. Figure 2 In D, the significant reduction in biofilm at 1×MIC concentration was also due to the significant inhibition of bacterial growth by SC-43 at 1×MIC concentration; while 1 / 8×MIC and 1 / 4×MIC concentrations, although not significantly inhibiting bacterial growth, did reduce biofilm formation in CHS791; SC-43 at 1 / 2×MIC concentration also significantly reduced biofilm formation in YUSA218 and CHS791. Therefore, SC-43 can reduce biofilm formation in two ways: one is by inhibiting bacterial growth, thereby reducing biofilm production; the other is by inhibiting and reducing biofilm formation without affecting bacterial growth.

[0052] Example 4

[0053] Experiment on the bactericidal activity of SC-43 against Staphylococcus aureus.

[0054] Two models were used to investigate the bactericidal activity of SC-43 against Staphylococcus aureus MSSA (SA113) and MRSA (YUSA145). One model involved adding different concentrations of SC-43 and a control antibiotic during the logarithmic growth phase. The other model involved culturing to the plateau phase to simulate a persistent bacterial model, then adding SC-43 and a control antibiotic, and continuing culturing for 5 days. Specific steps included:

[0055] For the sterilization curve in the logarithmic phase, firstly, a single clone was picked and added to 3 ml of TSB medium and shaken overnight. It was then diluted 1:200 with TSB and cultured for 3 h to reach the logarithmic phase, for a total of 20 ml. After thorough mixing, it was divided into shaking tubes, 4 ml per tube, and SC-43 at 4×MIC and 8×MIC concentrations, vancomycin Van (8×MIC concentration, 8 μg / ml), and linezolid Lzd (8×MIC concentration, 16 μg / ml) were added respectively. The tubes were then shaken and cultured at 37℃. At 0 h, 3 h, 6 h, 8 h, and 24 h, 100 μl samples were taken and added to 900 μl of physiological saline for serial dilution. The samples were then plated and cultured for 24 h before counting. For the plateau phase sterilization curve, fresh overnight shaken SA113 and YUSA145 bacterial suspensions were used and shaken 1:200 in CAMHB for 14-16 hours, totaling 20 ml. After thorough mixing, the mixture was divided into 5 ml portions of 25 ml Erlenmeyer flasks, and SC-43 at concentrations of 4×MIC and 8×MIC, and vancomycin Van (8 μg / ml) at concentration of 8×MIC were added to each flask. The flasks were then incubated at 37°C. At 0 h, 24 h, 48 h, 96 h, and 120 h, 100 μl samples were taken and added to 900 μl of physiological saline for serial dilution. The samples were then plated and incubated for 24 hours before counting.

[0056] The results are as follows Figure 3 As shown, in the logarithmic phase model of strain SA113, 4×MIC and 8×MIC SC-43 reduced the bacterial load to 188-fold and 477-fold, respectively, within 3 hours, demonstrating bactericidal effects superior to the control groups of vancomycin (Van) and linezolid (Lzd) by 67-fold and 17-fold, respectively. After 6 hours of treatment, 4×MIC SC-43 and 8×MIC SC-43 reduced the bacterial load by more than 1,100-fold, while the Lzd group only reduced it by 39-fold. After 8 hours of treatment, 8×MIC SC-43 and 8×MIC Van reduced the bacterial load from 10% of the control group. 9 The order of magnitude decreased to 10 5 4×MIC SC-43 can reduce bacterial count to 10. 5-6 The order of magnitude, while 8×MIC Lzd can reduce SA113 strain to 10 in 8 hours. 6Within 24 hours, due to drug metabolism, the bacterial count slightly increased, but the bactericidal effect in each group could still reduce the bacterial count from 10... 9 Reduced to 10 5-6 For the YUSA145 strain, in the logarithmic phase model, after 3 hours of drug treatment, 8×MICSC-43 reduced the bacterial count by 185-fold, significantly higher than the Van (9-fold) and Lzd groups (15-fold). After 6 hours, 8×MICSC-43 continued to exert its bactericidal effect, reducing the bacterial count by 1811-fold. The bactericidal effect also improved in the Van and Lzd groups, with viable bacterial counts decreasing by 294-fold and 454-fold, respectively. There were no significant differences between the groups at 8 hours and 24 hours of drug treatment. Therefore, in the logarithmic phase model, after 24 hours of treatment, due to drug metabolism, the bactericidal effect is similar to that of other first-line antibiotic control groups. However, SC-43 exhibits higher bactericidal activity in the early stages, which is an advantage.

[0057] In the plateau phase model, for strain SA113, SC-43 treatment for 24 hours only reduced the bacterial load from 10... 9 Reduced to 10 8 After 48 hours of treatment, 4×MIC SC-43 and 8×MIC SC-43 reduced the bacterial count from 10... 9 Reduced to 10 5 and 10 4 Order of magnitude; after 72 hours of treatment, 4×MIC SC-43 and 8×MIC SC-43 reduced the bacterial count from 10... 9 Reduced to 10 3-4 and 10 2 Order of magnitude; surprisingly, after 96 hours, 4×MIC SC-43 and 8×MIC SC-43 completely killed the bacteria, while the control group had little effect on killing bacteria remaining in the plateau phase. For MRSA strains, SC-43 also demonstrated high bactericidal activity and ability: after 24 hours of SC-43 treatment, the bacterial count was reduced from 10... 9 Reduced to 10 8 After 48 hours of treatment, 4×MIC SC-43 and 8×MIC SC-43 reduced the bacterial count from 10... 9 Reduced to 10 7 and 10 4-5 Order of magnitude; after 72 hours of treatment, 4×MIC SC-43 and 8×MIC SC-43 reduced the bacterial count from 10... 9 Reduced to 10 5 and 10 3 Order of magnitude; after 96 h and 120 h of treatment, 4×MIC SC-43 and 8×MIC SC-43 reduced the bacterial count from 10... 9 Reduced to 10 3 and 10 2The number of bacteria was reduced to a few hundred to a little over a thousand.

[0058] In summary, SC-43 showed superior bactericidal efficacy against bacteria in the logarithmic growth phase compared to Van and Lzd in the first 6 hours, while its efficacy was similar to that of the control antibiotics after 24 hours. For bacteria simulating persistent bacteria in the plateau phase, SC-43 demonstrated significantly higher bactericidal efficacy than vancomycin, eradicating all MSSA strains and reducing MRSA strains to only a few hundred bacteria. Therefore, SC-43 exhibits highly effective efficacy against Staphylococcus aureus.

[0059] Example 5

[0060] Safety tests of SC-43.

[0061] Numerous studies and clinical cases have shown that high-potency antibiotics have greater side effects on the human body than ordinary antibiotics, and are more likely to cause more serious chain reactions, such as liver and kidney failure and damage to the immune system. Therefore, highly safe antibiotics are a necessary condition for the development of new antibacterial drugs. To test the drug toxicity of SC-43, this embodiment uses CCK-8 and hemolysis assays for cytotoxicity detection. Specific steps include:

[0062] For the CCK-8 assay, suspension or adherent target cells were seeded into 96-well plates, 12,500 cells per well. The diluted test drug was added immediately to suspension cells, while adherent cells were allowed to adhere for 24 hours before addition. A blank control group without the drug was also included. The plates were incubated at 37°C and 5% CO2 for 24 hours. 10 μl of CCK8 (MCE) solution was added to each well, and the cells were incubated for another 1.5 hours. The absorbance at 450 nm (OD450) was then measured. The cytotoxicity of the drug was calculated based on the absorbance values ​​using the formula: Survival rate (%) = (Experimental group absorbance - Blank group absorbance) / (Control group absorbance - Blank group absorbance) * 100%. For the hemolysis assay, different concentrations of SC-43 were added to PBS containing blood cells, incubated at 37°C for 1 hour, centrifuged, and the supernatant was collected. The absorbance was measured at OD540 nm. Triton X-100 was used as a positive control, and PBS was used as a negative control. The hemolysis rate was calculated as: experimental group - negative control group / positive control group - negative control group * 100%.

[0063] The results are as follows Figure 4As shown, the minimum concentrations (CC50) of SC-43 required to kill 50% of the host cells in human non-small cell lung cancer cells A549, human liver cancer cells HepG2, human hepatic stellate cells LX-2, mouse mononuclear macrophages J774A.1, human embryonic kidney cells 293T, and normal human bronchial epithelial cells BEAS-2B were 9.162 μM, 15.31 μM, 7.255 μM, 8.97 μM, 6.32 μM, and 23.51 μM, respectively. The results indicated that SC-43 exhibited relatively low toxicity within the normal MIC working concentration range against Gram-positive bacteria (MIC range of 0.39-3.125 μM for 106 bacterial strains, of which 93 strains were 0.39-0.78 μM, 11 strains were 1.56 μM, and 2 strains were 3.125 μM). Figure 4 AF).

[0064] For the hemolysis test, the results are as follows: Figure 4 GH, as can be seen, SC-43 shows high safety, does not cause hemolysis, and only causes very weak hemolysis that is not visible to the naked eye at high concentrations of 50-200 μM.

[0065] Therefore, from a safety perspective, the SC-43 has research and application value for further development, modification, and application.

[0066] Example 6

[0067] Research experiments on the antibacterial effect of SC-43 targeting the cell membrane.

[0068] In the process of screening antimicrobial drugs and studying their mechanisms, experiments have revealed that many drugs target the cell membrane. Therefore, changes in membrane permeability were detected by measuring the fluorescence value of propidium iodide (PI) after SC-43 treatment. Furthermore, the effects of adding additional phospholipids on their antimicrobial activity were investigated. The specific steps of these two experiments included:

[0069] Membrane permeability test: Overnight revived Staphylococcus aureus strain YUSA145 was re-inoculated and shaken to the logarithmic phase. It was washed twice with physiological saline and resuspended in 10 times the initial volume of physiological saline. 200 μl of the resuspension was added to each well of a black opaque 96-well plate. DIBAC4(3) or PI was added to the resuspension to a concentration of 1 μM. High-content fluorescence intensity was measured for 10 min, with a 2-min interval between tests. The plate was then removed, and the corresponding concentration of the prepared drug was quickly added, with 0.1% Triton as a positive control and DMSO as a negative control. High-content fluorescence intensity was measured up to 70 min, with a 2-min interval between tests. N = 3. Values ​​are Mean ± SEM.

[0070] Experiment on the effect of phospholipids on MIC: In a 96-well plate, an excess of cultured bacterial solution (1:500 dilution) was added, followed by the diluted phospholipids and drugs. The plate was incubated for 18-24 hours, and the changes in the minimum inhibitory concentration were observed.

[0071] The results are as follows Figure 5 As shown, although the increase in fluorescence value of strains SA113 and YUSA145 after treatment with SC-43 was not as large as that after treatment with 0.1% Triton X-100 in the positive control group, an increase in PI fluorescence was observed. Furthermore, the results for strain YUSA145 showed a concentration-dependent fluorescence increase trend. These results reveal that SC-43 can induce changes in membrane permeability. Figure 5 AB). A combined phospholipid assay was conducted to investigate whether treatment of SC-43 with diphosphatidylglycerol (cardiolipin CL), phosphatidylcholine (lecithin PC), phosphatidylethanolamine (cephalin PE), and phosphatidylglycerol (PG) altered the minimum inhibitory concentration (MIC) of SC-43 against SA113, YUSA145, and Enterococcus faecalis EF16C51. The results showed that the addition of all four phospholipids increased the MIC of SC-43 against the strains and decreased its antibacterial effect, exhibiting a concentration-dependent effect. This was particularly evident against Staphylococcus aureus SA113 and YUSA145; at a phospholipid concentration of 2 μg / ml, the MIC of SC-43 increased twofold, and at a concentration of 128 μg / ml, the MIC increased 16-32 times, significantly reducing its antibacterial effect. Figure 5 CD). For Enterococcus faecalis, at a concentration of 128 μg / ml, the MIC of SC-43 increased 4-8 times. Although the decrease was not as large as that for Staphylococcus aureus, it still significantly reduced its antibacterial effect. Figure 5 E). This shows that, to a certain extent, the SC-43 targeted membrane exerts an antibacterial effect.

[0072] Furthermore, to further determine whether SC-43 can target the bacterial cell membrane to exert its antibacterial effect, we examined the membrane changes of the SC-43-treated strains 2 hours after treatment using electron microscopy. The results showed that both 4×MIC and 8×MIC concentrations could rupture the membrane, leading to bacterial death, and the higher the concentration, the greater the bacterial death. Figure 6 (AB). Through the above experiments, it can be concluded that the mechanism by which SC-43 has highly efficient antibacterial activity includes targeting the cell membrane.

[0073] Example 7

[0074] Proteomic target analysis of SC-43 against Staphylococcus aureus.

[0075] To further investigate the mechanism of SC-43's high antibacterial efficiency, proteomics was first used. YUSA145 strain was treated with SC-43 at 1 / 2×MIC for 2 hours, and the bacterial cells were collected for proteomics sequencing.

[0076] The specific steps include: treating Staphylococcus aureus YUSA145 with SC-43 at 1 / 2×MIC for 2 hours, collecting the bacterial cells, lysing them, extracting proteins, and analyzing the protein expression levels using a high-resolution mass spectrometer (QE).

[0077] The results are as follows Figure 7 and Figure 8 As shown, using a 1.2-fold differential protein cutoff, 40 proteins were upregulated and 54 proteins were downregulated. Figure 7 (AB), some significant proteins are labeled in the volcano plot. Biological process analysis was performed on these differentially expressed proteins. Figure 7 C) Enrichment was found primarily in nuclebase metabolic processes, tetrahydrofolate interconversion, and one-carbon metabolic processes; KEGG enrichment analysis revealed only one one-carbon pool of byfolate. Figure 8 A); Molecular functional enrichment analysis revealed that the main pathways included methylenetetrahydrofolate cyclohydrolase activity, methylenetetrahydrofolate-ehydrogenase (NADP+) activity, and other pathways. Figure 8B); For cellular component enrichment analysis, the results showed that the components were mainly concentrated in the envelope, outer membrane-bound periplasmic space, cell envelope, ATP-binding cassette transporter complex, ATPase-dependent transmembrane transport complex, transmembrane transport complex, transport complex, and plasma membrane protein complex. Figure 8 C) Gene analysis of the above-mentioned cellular components revealed that differentially expressed protein genes related to structural pathways in the periplasmic space, cell membrane, and transport complex were all within 3% ( Figure 8 D); Protein-protein interaction analysis (PPI) revealed a significant one-carbon pool by folate, with high expression of proteins from four related genes (folD, gcvT, fhs, and purN). Specific information on upregulated and downregulated proteins is shown in Tables 4 and 5, recording protein name, protein family, gene name, log2 foldchange value, P-value, transmembrane protein, subcellular localization, and signaling pathway. Of the 40 upregulated proteins, 4 were related to the membrane and cell wall; of the 54 downregulated proteins, 19 were related to the membrane and 1 to the cell wall. Therefore, a total of 25.5% of the differentially expressed proteins were related to the membrane and cell wall.

[0078] In summary, the proteomic analysis of SC-43 further reveals that the mechanism by which SC-43 exerts its antibacterial effect is related to targeting the cell membrane.

[0079] Table 4-1 Upregulated protein 1

[0080]

[0081] Table 4-2 Upregulated Protein II

[0082]

[0083] Table 5-1 Downregulated protein 1

[0084]

[0085] Table 5-2 Downregulated protein II

[0086]

[0087] Example 8

[0088] Whole-genome sequencing analysis of SC-43-induced drug resistance.

[0089] To further confirm the target of SC-43, a method of inducing resistance followed by whole-genome sequencing was used to detect the target. Gram-positive bacteria were continuously induced in vitro with sub-inhibitory concentrations of SC-43 for different generations until the MIC concentration increased at least fourfold. This induced resistance mutations, and whole-genome sequencing was then performed to analyze the mutated genes. Based on the function of the mutated genes, the potential antibacterial mechanism of SC-43 was analyzed. Gene knockout or reversion strains were then constructed based on the mutated genes, and the antibacterial phenotype of SC-43 was examined.

[0090] In this embodiment, three bacterial strains, YUSA145, SA113, and EF16C51, were used for induction, reaching generations 43, 41, and 44, respectively. SC-43 was induced to generations 25, 23, and 26 using concentrations of 1×MIC, 1×MIC, and 2×MIC. Currently, the MIC after induction has only increased by 2-fold, as shown in Table 6. Therefore, induction will continue. Some drugs easily induce resistance, with highly resistant strains appearing after ten or twenty generations. The current progress in inducing SC-43 suggests, to some extent, that SC-43 may not easily lead to bacterial resistance.

[0091] Table 6 Progress of Drug-Induced Resistance to SC-43

[0092]

[0093] All experiments in the above embodiments used GraphPad Prism 8.0 software for data processing and image rendering. P < 0.05 was considered statistically significant.

[0094] 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 application of SC-43 in the preparation of drugs for treating Gram-positive bacterial infections, characterized in that: The SC-43, CAS number 1400989-25-4, has the function of inhibiting the growth of Gram-positive bacteria and biofilm formation; the Gram-positive bacteria are at least one of Enterococcus faecalis, Enterococcus faecium, Streptococcus agalactiae, Staphylococcus hominis, Staphylococcus hemolyticus, and Staphylococcus capitulata.

2. The use of SC-43 according to claim 1 in the preparation of drugs against Gram-positive bacterial infections, characterized in that: The concentration of SC-43 in the treatment system is not less than 0.39 μM.

3. The use of SC-43 according to claim 1 in the preparation of drugs against Gram-positive bacterial infections, characterized in that: The drug is an injection, tablet, pill, capsule, suspension, granule, spray, or emulsion.

4. The application of SC-43 in the preparation of coatings that inhibit Gram-positive bacteria, characterized in that: The coating is used on the surface of medical devices. The CAS number of SC-43 is 1400989-25-4. SC-43 has the function of inhibiting the growth of Gram-positive bacteria and the formation of biofilms. The Gram-positive bacteria are at least one of Enterococcus faecalis, Enterococcus faecium, Streptococcus agalactiae, Staphylococcus hominis, Staphylococcus hemolyticus, and Staphylococcus capitella.

5. The application of SC-43 according to claim 4 in the preparation of coatings that inhibit Gram-positive bacteria, characterized in that: In the coating, the concentration of SC-43 is not less than 0.39 μM.

6. The application of SC-43 in the preparation of antibacterial agents against Gram-positive bacteria, characterized in that: The CAS number of SC-43 is 1400989-25-4. SC-43 has the function of inhibiting the growth of Gram-positive bacteria and biofilm formation. The Gram-positive bacteria are at least one of Enterococcus faecalis, Enterococcus faecium, Streptococcus agalactiae, Staphylococcus hominis, Staphylococcus hemolyticus, and Staphylococcus capitulata.

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