Application of PFM01 in preparation of gram-positive bacterial infection resisting medicine

Abstract

The invention provides an application of PFM01 in preparation of a medicine for resisting gram-positive bacterial infection, and the CAS number of the PFM01 is 1558598-41-6. According to the technical scheme, the novel medical application of PFM01 is disclosed, the PFM01 has good antibacterial activity and anti-biofilm activity on various gram-positive bacteria, can inhibit growth of staphylococcus aureus, staphylococcus epidermidis, enterococcus faecalis, enterococcus faecium and the like, especially has an obvious inhibiting effect on methicillin-resistant staphylococcus aureus (MRSA), and can be used for preparing the medicine for preventing and treating the diseases such as the staphylococcus aureus, the staphylococcus epidermidis, the enterococcus faecalis, the enterococcus faecium and the like. And compared with vancomycin, the vancomycin compound shows more obvious bactericidal activity, and can obviously inhibit the formation of a biofilm and kill bacteria in the formed biofilm.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of PFM01 in preparation of a drug for resisting gram-positive bacterial infection. BACKGROUND

[0002] Gram-positive bacteria, such as Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus, are important pathogens commonly seen in clinic, which can cause skin and soft tissue infection, pneumonia, bacteremia and a series of diseases related to implant infection, and pose a serious threat to public health.

[0003] Currently, the treatment of such bacterial infection is facing two severe challenges:

[0004] First, the problem of drug resistance is increasingly prominent. With the widespread use of antibiotics, the emergence and spread of multi-drug resistant strains have become a global health crisis. Among them, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) are particularly troublesome. MRSA, through the acquisition of the mecA gene encoding PBP2a protein, is resistant to almost all beta-lactam antibiotics, and its high morbidity and mortality are listed as a high-priority pathogen by WHO. In addition, bacteria also develop resistance to new antibiotics such as daptomycin and linezolid through complex mechanisms such as target mutation and efflux pump activation, leading to a decreasing number of available drugs in clinic and a treatment dilemma.

[0005] Second, the formation of biofilm greatly increases the difficulty of treatment. More than 80% of chronic infections are related to biofilm. Bacteria form a protective membrane structure wrapped in polysaccharide matrix, which significantly hinders the penetration of antibiotics and induces bacterial metabolic dormancy, making them more than 100 times more resistant. On the other hand, it helps bacteria to escape from host immune clearance and provides convenience for drug-resistant gene exchange. This is very common in medical device-related infections, and conventional drug treatment often fails, often requiring surgical intervention, which greatly increases the medical burden.

[0006] Therefore, the development of new antibacterial drugs with a completely new mechanism of action that can effectively overcome drug resistance and eliminate biofilm is an urgent need to be solved in current clinical practice. SUMMARY

[0007] In view of the above technical problems, the application discloses application of PFM01 in preparation of a drug for resisting gram-positive bacterial infection. PFM01 shows a broad-spectrum growth inhibition effect on various gram-positive bacteria, especially on Staphylococcus aureus, and shows antibacterial activity and bactericidal activity, and can inhibit the formation of Staphylococcus aureus biofilm, and can also kill bacteria in the biofilm, thereby providing a reference for the development of antibacterial drugs based on PFM01 as a lead structure.

[0008] To this end, the technical solution adopted by the application is that:

[0009] Use of PFM01 in the preparation of a drug for resisting gram-positive bacterial infection, wherein the PFM01 has a CAS number of 1558598-41-6.

[0010] The PFM01 has a structural formula as shown in formula (1):

[0011]

[0012] PFM01 is a small molecule compound, which is an N-alkylated derivative of Mirin, and mainly serves as a selective inhibitor of MRE11 endonuclease. MRE11 is a core component of the MRN complex (MRE11-RAD50-NBS1), which plays a key role in the detection and repair of DNA double-strand breaks (DSBs). PFM01 interferes with the end-resection process of DSBs by specifically inhibiting the endonuclease activity of MRE11 (without affecting its exonuclease activity), thereby promoting the non-homologous end joining (NHEJ) repair pathway and reducing the activity of the homologous recombination (HR) pathway. Studies have shown that PFM01 can enhance the sensitivity of tumor cells to radiotherapy or chemotherapy, because inhibiting MRE11 can weaken the DNA repair ability of cancer cells, leading to DNA damage accumulation and cell death. For example, in a breast cancer model, inhibition of MRE11 can inhibit tumorigenesis by regulating cGAS (cyclic GMP-AMP synthase) activation. In addition, PFM01 shows potential in reducing chromosomal breaks and regulating DSB repair balance in in vitro experiments, and can be used as an adjuvant drug for the treatment of diseases related to DNA damage response defects, such as certain genetic cancer syndromes. However, the antibacterial activity of PFM01 has not been reported.

[0013] However, the present inventors have found through a large number of experiments that PFM01 exhibits good antibacterial activity against gram-positive bacteria and can significantly inhibit the formation of Staphylococcus aureus biofilm. Moreover, PFM01 exhibits more significant bactericidal activity than vancomycin. In addition, the formation of Staphylococcus aureus biofilm is an important virulence factor leading to chronic infection, and only a few traditional antibiotics are effective against biofilm. PFM01 has a significant inhibitory effect on the formation of biofilm of some clinical Staphylococcus aureus, and has less toxicity to cells, and the half-inhibitory concentration (IC 50 ) of PFM01 on the proliferation of human renal epithelial cell line 293T and mononuclear macrophage J774A.1 is significantly higher than the bacteriostatic MIC. These results indicate that PFM01 has potential application value in the treatment of clinical gram-positive bacterial infection.

[0014] As a further improvement of the present application, the gram-positive bacteria are at least one of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and Enterococcus faecium.

[0015] As a further improvement of the present application, the concentration of the PFM01 in the processing system is not less than 0.92 μg / ml.

[0016] As a further improvement of the present application, the medicine is a pharmaceutical composition or preparation. Further, the medicine is an injection, a tablet, a pill, a capsule, a suspension, a granule, a spray or an emulsion.

[0017] The present application also discloses the use of PFM01 in preparing a coating for inhibiting gram-positive bacteria, the coating being used for the surface of a medical instrument, the CAS number of the PFM01 being 1558598-41-6 and the structural formula being shown as formula (1). The coating has good bacteriostatic or bactericidal effect on gram-positive bacteria including Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium and the like.

[0018] As a further improvement of the present application, the concentration of the PFM01 in the coating is not less than 0.92 μg / ml.

[0019] The present application also discloses the use of PFM01 in preparing a disinfectant for inhibiting gram-positive bacteria, the CAS number of the PFM01 being 1558598-41-6 and the structural formula being shown as formula (1); the disinfectant has good bacteriostatic or bactericidal effect on gram-positive bacteria including Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium and the like.

[0020] As a further improvement of the present application, the concentration of the PFM01 in the coating is not less than 0.92 μg / ml.

[0021] Compared with the prior art, the present application has the beneficial effects that:

[0022] The technical scheme of the present application discloses a new medical use of PFM01, PFM01 has good antibacterial activity on various gram-positive bacteria, especially has obvious inhibitory effect on Staphylococcus aureus, and shows more significant bactericidal activity than vancomycin, and can significantly inhibit the formation of biofilm. In addition, PFM01 has bactericidal effect on Staphylococcus aureus. The toxicity of PFM01 on cells is small at the MIC dose. These results suggest that PFM01 has the possibility of treating Staphylococcus aureus infection. Moreover, the MIC of PFM01 is equivalent to that of the commonly used clinical antibiotics, and is suitable for clinical use. It can be seen that PFM01 has potential application value in the clinical treatment of Staphylococcus aureus infection. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1Growth curves of different Staphylococcus aureus after adding PFM01 in the embodiment of the present application, wherein (a)-(c) are Staphylococcus aureus ATCC29213, SA113, and YUSA145, respectively.

[0024] Figure 2 Bactericidal curve analysis of Staphylococcus aureus MSSA strain (SA113) and MRSA strain (YUSA145) by PFM01 in the embodiment of the present application, wherein (a) is SA113, and (b) is YUSA145.

[0025] Figure 3 Biofilm activity analysis of Staphylococcus aureus by PFM01 in the embodiment of the present application, wherein (a) is the inhibition effect of different sub-inhibitory concentrations of PFM01 on biofilm formation of MSSA strain (SA113) and MRSA strain (YUSA145), and (b) is the killing activity analysis of different concentrations of PFM01 on bacteria in mature biofilm of SA113 and YUSA145.

[0026] Figure 4 Effect curve of PFM01 on cell membrane permeability of Staphylococcus aureus YUSA145 in the embodiment of the present application.

[0027] Figure 5 PFM01 bacteriostatic activity analysis of different bacterial membrane phospholipid components in the embodiment of the present application, wherein MIC represents the minimum inhibitory concentration, CL represents cardiolipin, PG represents phosphatidylglycerol, and PE represents phosphatidylethanolamine.

[0028] Figure 6 PFM01 proliferation toxicity detection results on 293T and J774A.1 cells in the embodiment of the present application, wherein (a) is 293T cells, and (b) is J774A.1 cells. DETAILED DESCRIPTION

[0029] The preferred embodiments of the present application are further described in detail below.

[0030] Gram-positive bacteria used in the following examples, including 48 strains of MRSA, 32 strains of MSSA, 22 strains of Staphylococcus epidermidis, 19 strains of Enterococcus faecium and 20 strains of Enterococcus faecalis clinical strains were collected from different inpatients in the hospital. All clinical strains were identified by Phoenix 100 automatic microbial identification system, and all strains were re-identified by matrix-assisted laser desorption ionization / time-of-flight mass spectrometry (MALDI-TOF-MS) after subculture. Quality control strains Staphylococcus aureus SA113 (ATCC35556), ATCC29213 and Enterococcus faecalis ATCC29212 were purchased from ATCC strain library.

[0031] Example 1

[0032] In this example, the effect of PFM01 on the growth of Staphylococcus aureus and Enterococcus faecalis standard strains was detected by 96-well plate high-throughput. The specific steps are as follows:

[0033] Take the overnight culture of bacteria (Staphylococcus aureus ATCC35556, ATCC29213 and Enterococcus faecalis ATCC29212) and adjust the turbidity to 0.5 McFarland (bacterial amount is about 1.0-1.5×108cfu / mL) with a turbidity cup. Take the bacterial solution and dilute it 1:200 with CAMHB medium, then add it to a 96-well plate, 12 wells per row, 200uL per well. Add PFM01 diluted to 100uM, add 200uL CAMHB medium to the 12th well as negative control. Take compounds RCGD423, Fluzoparib, Fluorizoline, ELN484228, SORT-PGRN interaction inhibitor 1, JNJ-63576253, IV209, UBCS039, EB-3D, MLT-748, CLK-IN-T3, MPO-IN-28, 7-BIA, Jarin-1, NAE-IN-M22, K-Ras-PDEδ-IN-1, GW406108X, MALAT1-IN-1, KS15, ILK-IN-3, TXNIP-IN-1, (S)-TXNIP-IN-1, Glumetinib, RMC-4550, SRS11-92, VPC-14449, PIN1 inhibitor API-1, ML401 as control samples, observe the results after incubation at 37℃ for 24 hours. The compounds added in the wells where no bacteria growth can be seen by naked eye are considered to have potential antibacterial activity.

[0034] Results: It was found that only the Staphylococcus aureus ATCC29213, ATCC35556, ATCC29213 and Enterococcus faecalis ATCC29212 added with 100 uM PFM01 showed clear culture solution after 24 hours of culture, and no bacteria growth was observed, and the absorbance at 600 nm (OD600) was less than 0.1. It can be seen that PFM01 has potential antibacterial activity on the growth of Staphylococcus aureus and Enterococcus faecalis.

[0035] Further, the minimum inhibitory concentration (MIC) of PFM01 on various common gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium) was detected by micro-broth dilution method. The specific steps are as follows:

[0036] The overnight culture solution was adjusted to a turbidity of 0.5 McFarland (bacterial amount of about 1.0-1.5 x 10 8 The bacterial solution was diluted 1:100 with CAMHB medium and added to a 96-well plate, 12 wells per row, 10 gradient wells (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39 uM) of drugs were set, 200 uL of the above bacterial solution was added to the 11th well as a positive control, and 200 uL of CAMHB medium was added to the 12th well as a negative control. The MIC value determination culture conditions and time of each antibacterial drug were carried out according to the CLSI guide, and the results were observed after 18 hours of culture at 37°C. The drug concentration with no visible bacterial growth by naked eye, i.e. clear culture solution, was taken as the minimum inhibitory concentration MIC value.

[0037] The MIC value distribution results of PFM01 on Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and Enterococcus faecium are shown in Table 1. It can be seen that PFM01 has better antibacterial activity on various gram-positive bacteria, and the MIC value mainly distributes between 6.25 uM (≈1.83 ug / mL) and 12.5 uM (≈3.67 ug / mL), among which the inhibition effect on Staphylococcus epidermidis is the best, and the MIC 50 is 6.25 uM.

[0038] Table 1 Drug sensitivity of common clinical hospital infection gram-positive bacteria to PFM01

[0039] Note: S. aureus: Staphylococcus aureus; E. faecalis: Enterococcus faecalis; S. epidermidis: Staphylococcus epidermidis; E. faecium: Enterococcus faecium; n is the number of strains measured.

[0040] Example 2

[0041] PFM01 effect on the growth of S. aureus.

[0042] In order to verify whether PFM01 can inhibit the growth of S. aureus, different concentrations of PFM01 were used to treat S. aureus standard strains ATCC29213, SA113 and MRSA clinical strain YUSA145, and the OD 600 The value represents the growth of bacteria. The specific steps are as follows: take the overnight culture liquid (S. aureus ATCC29213, SA113 and YUSA145) and dilute 1000 times with TSB medium, then add different concentrations (1 / 4x, 1 / 2x and 1x) of PFM01 to a 96-well plate, and then place it in a full-automatic growth curve analyzer. The absorbance (OD600) at 600 nm wavelength was measured every 1 hour to detect the content of planktonic bacteria in the culture supernatant. The incubation temperature was 37°C, and the growth curve was drawn.

[0043] The growth curve analysis is shown in Figure 1 As can be seen, at 1 / 2x MIC concentration, PFM01 can inhibit the growth of S. aureus planktonic bacteria, but does not affect the maximum growth level. When the concentration reaches 1x MIC, the growth of S. aureus ATCC29213, SA113 and YUSA145 is completely inhibited. These results preliminarily indicate that PFM01 has the potential to be used as an anti-S. aureus infection drug.

[0044] Example 3

[0045] PFM01 effect on the growth of S. aureus.

[0046] In this example, the time and dose-dependent effects of PFM01 on the antibacterial activity of gram-positive bacteria were studied, and the activity was compared with that of the clinical antibiotic vancomycin. The specific steps include:

[0047] The logarithmic phase (OD 600 = 0.5) MRSA clinical strain YUSA145 bacterial liquid and standard strain SA113 stable phase culture liquid were diluted 100 times, respectively, and then incubated with 2x MIC (25 μM), 4x MIC (50 μM) PFM01 and 4x MIC vancomycin in a shaking bed at 200 rpm. Subsequently, samples were collected at 0, 3, 6, and 24 hours, respectively, and serially diluted with sterile saline, and then plated on TSB plates and incubated at 37°C. After 24 hours, the colonies were counted. The colony count was expressed as colony forming units per milliliter (CFU / mL).

[0048] The killing curve was plotted with the killing time as the abscissa and the logarithm of the colony forming units per milliliter as the ordinate. The killing curve analysis is shown inFigure 2 As shown, the concentration of PFM01 can be observed to have bactericidal effect on both S. aureus YUSA145 and SA113 at 2xMIC, and the bactericidal effect is better at 4xMIC. For MRSA strain YUSA145, the bacterial count is reduced to the detection limit within 24 hours after treatment with 4xMIC PFM01, and the bactericidal effect is stronger than that of 4xMIC vancomycin.

[0049] Example 4

[0050] In this example, the crystal violet method was used to study the antibiofilm activity of PFM01 on S. aureus strains. The specific steps were as follows:

[0051] The crystal violet staining method was used to detect the change in biofilm amount, and each test was repeated 3 times with 3 replicate wells, and the average value was taken as the final test result. Specifically, the overnight MRSA clinical strain YUSA145 and standard strain SA113 bacterial liquid were diluted 1000 times with TSBG medium containing 2% glucose in a 96-well plate, 3 replicate wells per group, and different sub-inhibitory concentrations of PFM01 were added. The blank medium without bacterial liquid was used as the negative control, and the solvent DMSO was used as the positive control. After incubation in a 37°C incubator for 24 hours, the culture solution was gently aspirated and washed with sterile PBS for 3 times, and then air-dried at room temperature. Methanol fixation for 15 minutes, 1% crystal violet staining for 15 minutes, washing with sterile water for 3 times until the control wells were colorless, and air-dried at room temperature. 200 μL of anhydrous ethanol was added to each well, shaken for 1 minute, and the absorbance at 570 nm was measured on a microplate reader.

[0052] In addition, the CFU bacterial count method was used to detect the effect of PFM01 on the amount of bacteria in the biofilm: the overnight MRSA clinical strain YUSA145 and standard strain SA113 bacterial liquid were diluted 1000 times with TSBG medium containing 2% glucose in a 96-well plate, 3 replicate wells per group, and mature biofilm of S. aureus was formed overnight. Then different concentrations (50 μM, 100 μM or 200 μM) of PFM01 were added to the biofilm culture solution, and the solvent DMSO was used as the positive control. After further incubation in a 37°C incubator for 24 hours, the culture solution was gently aspirated and washed with sterile PBS for 3 times. The biofilm was blown off by aspirating sterile water. Then, the biofilm bacteria were serially diluted with sterile normal saline, and plated on TSB plates for incubation at 37°C. After 24 hours, the colony count was performed. The colony count was expressed in colony forming units per milliliter (CFU / mL).

[0053] The results of semi-quantitative analysis of biofilm by crystal violet staining are shown in Table 2. Figure 3As shown in the figure, when PFM01 is 1 / 8 x MIC, the biofilm biomass formed by YUSA145 and SA113 is reduced. When the concentration of PFM01 is 1 / 2 x MIC, the biofilm formation of YUSA145 and standard strain SA113 is inhibited by more than 50%. In addition, the results of bacterial counting CFU in biofilm show that when the concentration of PFM01 is 100 μM, it can significantly reduce the number of viable bacteria in the biofilm, and when the concentration of PFM01 is 200 μM, it can reduce the number of viable bacteria of Staphylococcus aureus YUSA145 and SA113 by more than 99% (> 2log10). It shows that PFM01 can inhibit the formation of Staphylococcus aureus biofilm, and has a strong killing effect on bacteria in mature biofilm.

[0054] Example 5

[0055] This example is a study on the cell membrane permeability of PFM01 on Staphylococcus aureus YUSA145. The specific steps are as follows:

[0056] The logarithmic phase of gram-positive bacteria YUSA145 cells was adjusted to OD600 = 0.05, and 1 μM propidium iodide (PI) was incubated in 5 mM HEPES buffer (pH = 7.2) for 10 minutes in the dark. Then the suspension was treated with different concentrations of PFM01 (final concentration of 12.5 μM, 125 μM and 50 μM, corresponding to 1 x, 2 x and 4 x MIC), and 0.1% DMSO and 0.1% Triton were used as controls, and the fluorescence intensity (excitation wavelength 535 nm, emission wavelength 615 nm) was continuously monitored. PI is a membrane permeability cell nucleus staining reagent for detecting DNA, which releases fluorescence after embedding double-stranded DNA. PI cannot pass through the membrane of living cells, but can pass through the damaged cell membrane.

[0057] The monitoring results of fluorescence intensity are shown in the figure Figure 4 As shown in the figure, it is found that the fluorescence intensity of Staphylococcus aureus YUSA145 treated with PFM01 changes significantly, indicating that the cell membrane permeability of YUSA145 increases after treatment with PFM01, which shows that YUSA145 has damage effect on the cell membrane of Staphylococcus aureus.

[0058] Example 6

[0059] In this example, the effect of bacterial cell membrane phospholipid components on the antibacterial activity of PFM01 is studied. The specific steps include:

[0060] In this example, in order to analyze whether the antibacterial activity of PFM01 is related to the cell membrane, the chessboard micro-broth dilution method is used to detect the effect of different concentrations of bacterial cell membrane phospholipid components phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and cardiolipin (CL) on the minimum inhibitory concentration MIC of PFM01. The specific steps are as follows:

[0061] Take overnight Staphylococcus aureus MRSA clinical strain YUSA145 culture liquid and adjust the turbidity to 0.5 McFarland (bacterial amount is about 1.0-1.5x108CFU / mL) with a turbidity cup. Take the bacterial liquid and dilute it 1:100 with CAMHB medium, then add it to a 96-well plate, 12 wells per row, set 7 gradient wells for Etalocib (400, 200, 100, 50, 25, 12.5, 6.25 μM), add 200 μL of the above bacterial liquid to the 8th well as a positive control, and add 200 μL of CAMHB medium to the 9th well as a negative control; set 8 gradient wells for cell membrane phospholipid components (128, 64, 32, 16, 8, 4, 2, 1 μg / mL). The MIC value determination culture conditions and time are carried out according to the CLSI guidelines, and the results are observed after 18 hours of culture at 37°C. The drug concentration well in which the bacterial liquid precipitate cannot be seen by the naked eye is calculated as the MIC value.

[0062] The results obtained are plotted with different concentrations of phospholipids as the abscissa and the MIC concentration fold change of PFM01 as the ordinate, and the results are shown in Figure 5 It can be seen that the addition of bacterial cell membrane phospholipids, phosphatidylethanolamine (PE), to the culture medium has little effect on the antibacterial activity of PFM01, while phosphatidylglycerol (PG) and cardiolipin (CL) reduce the antibacterial activity of PFM01, causing a dose-dependent increase in the MIC of Staphylococcus aureus YUSA145. It can be seen that PFM01 may act on bacterial cell membrane phospholipids to exert antibacterial activity.

[0063] Example 7

[0064] This example is an experiment to study the effect of PFM01 on the proliferation toxicity of 293T and J774A.1 cells, and the specific steps are as follows:

[0065] Prepare 100 μL of 293T or J774A.1 cell suspension in a 96-well plate. Place the culture plate in the incubator for 24 hours of pre-culture (37°C, 5% CO2). Add 10 μL of PFM01 at different concentrations to the cell culture plate, setting 8 gradient wells (100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 μM). Incubate the culture plate in the incubator for 24 hours, and add 10 μL of CCK-8 solution to each well. Incubate the culture plate in the incubator for 1 hour. Measure the absorbance (OD450) at 450 nm with a microplate reader. Then calculate the cell viability by absorbance.

[0066] CCK-8 is a rapid high-sensitivity detection kit for cell proliferation and cytotoxicity based on WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazole monosodium salt). Its working principle is: in the presence of an electron coupling reagent, it can be reduced by dehydrogenase in mitochondria to generate a highly water-soluble orange yellow formazan product. The depth of color is proportional to the proliferation of cells and inversely proportional to cytotoxicity. The absorbance value (OD450) is measured at 450 nm wavelength using an enzyme-labeled instrument, which indirectly reflects the number of living cells. The results of PFM01 cytotoxicity on 293T and J774A.1 cells are shown in FIG. 1A and FIG. 1B, respectively. As shown in FIG. 1A and FIG. 1B, 25 mM PFM01 has no cytotoxicity on 293T or J774A.1 cells, and 50 mM PFM01 shows low inhibitory activity on 293T or J774A.1 cell proliferation. Overall, the IC50 of PFM01 on 293T and J774A.1 cell proliferation is 119.10 and 83.14 mM, which is higher than the MIC value of PFM01 on all tested gram-positive clinical strains, and has potential application potential for clinical gram-positive bacterial infection. Figure 6 As shown in FIG. 1A and FIG. 1B, 25 mM PFM01 has no cytotoxicity on 293T or J774A.1 cells, and 50 mM PFM01 shows low inhibitory activity on 293T or J774A.1 cell proliferation. Overall, the IC50 of PFM01 on 293T and J774A.1 cell proliferation is 119.10 and 83.14 mM, which is higher than the MIC value of PFM01 on all tested gram-positive clinical strains, and has potential application potential for clinical gram-positive bacterial infection.

[0067] All the above experiments use GraphPad Prism 8.0 software for data processing and drawing images. P<0.05 is considered to have statistical difference.

[0068] Comparative Example 1

[0069] In this comparative example, the minimum inhibitory concentration (MIC) of PFM01 structural analogs PFM39 (CAS No. 1310744-67-2) and Mirin (CAS No. 299953-00-7) on various gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium) was detected by micro-broth dilution method. The specific steps are as follows:

[0070] Take the overnight culture of bacteria and adjust the turbidity to 0.5 McFarland (bacterial amount is about 1.0-1.5x108cfu / mL) with a turbidity cup. Take the bacterial solution and dilute it 1:100 with CAMHB medium, then add it to a 96-well plate, 12 wells per row, set 10 gradient wells for drugs (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39 mM), add 200 uL of the above bacterial solution to the 11th well as positive control, and add 200 uL of CAMHB medium to the 12th well as negative control. After incubation at 37°C for 18 hours, the results are observed. The drug concentration well in which the bacterial precipitate cannot be seen with the naked eye is calculated as the MIC value.

[0071] In the present comparative example, the addition of different concentrations of PFM39 and Mirin did not inhibit the growth of the detected gram-positive bacteria, i.e. the MIC values of PFM39 and Mirin for S. aureus, S. epidermidis, E. faecalis and E. faecium were all greater than 200 μM. It can be seen that PFM39 and Mirin have no antibacterial activity against gram-positive bacteria, and the antibacterial activity of PFM01 against various gram-positive bacteria is structure-specific.

[0072] As can be seen from the results of the above examples, PFM01 exhibits better antibacterial activity against gram-positive bacteria, and the MIC values of PFM01 against common gram-positive bacteria causing hospital infections are mainly distributed in 6.25 μM (≈1.83 μg / mL) and 12.5 μM (≈3.67 μg / mL), and PFM01 can significantly inhibit the formation of biofilm of S. aureus clinical strains. In addition, PFM01 has stronger bactericidal effect against S. aureus than vancomycin, and the bacterial count of MRSA strain YUSA145 is reduced to the detection limit within 24 hours at a concentration of 4×MIC of PFM01. PFM01 at the MIC dose has no significant toxicity to 293T and J774A.1 cells, and PFM01 has inhibitory effect on hemolysis of S. aureus. These results suggest that PFM01 has the potential to treat S. aureus infection and is suitable for clinical use.

[0073] The above is a further detailed description of the present application in combination with specific preferred embodiments, and the specific implementation of the present application should not be limited to these descriptions. For ordinary skilled persons in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application, and all of them should be considered as falling within the protection scope of the present application.

Claims

1. The application of PFM01 in the preparation of drugs against Gram-positive bacterial infections, characterized in that: The PFM01 has a CAS number of 1558598-41-6; the Gram-positive bacteria are at least one of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium.

2. The application of PFM01 according to claim 1 in the preparation of drugs against Gram-positive bacterial infections, characterized in that: The concentration of PFM01 in the treatment system is not less than 0.92 μg / ml.

3. The application of PFM01 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 PFM01 in the preparation of coatings that inhibit Gram-positive bacteria, characterized in that: The coating is used on the surface of the medical device. The CAS number of the PFM01 is 1558598-41-6. The Gram-positive bacteria are at least one of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium.

5. The application of PFM01 according to claim 4 in the preparation of coatings that inhibit Gram-positive bacteria, characterized in that: In the coating, the concentration of PFM01 is not less than 0.92 μg / ml.

6. The application of PFM01 in the preparation of disinfectants that inhibit Gram-positive bacteria, characterized in that: The CAS number of PFM01 is 1558598-41-6, and the Gram-positive bacteria is at least one of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium.

7. The application of PFM01 according to claim 6 in the preparation of disinfectants that inhibit Gram-positive bacteria, characterized in that: In the disinfectant, the concentration of PFM01 is not less than 0.92 μg / ml.

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