Application of AZD-5991 in preparation of gram-positive bacterium infection resisting medicine

Abstract

The invention provides an application of AZD-5991 in preparation of a medicine for resisting gram-positive bacterium infection, and the AZD-5991 has a CAS number of 2143061 to 81 to 6. According to the technical scheme, the novel medical application of the AZD-5991 is disclosed, the AZD-5991 shows antibacterial activity on various gram-positive bacteria including staphylococcus aureus, staphylococcus epidermidis, enterococcus faecalis and enterococcus faecium, and can inhibit formation of staphylococcus aureus and enterococcus faecalis biofilms, so that the AZD-5991 can be used for preparing the antibacterial agent for preventing and treating the AZD-5991. Bacteria of a staphylococcus aureus mature biofilm can be killed, and floating growth and biofilm formation of staphylococcus aureus can be inhibited by destroying cell membranes and targeting FabI.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the use of AZD-5991 in the preparation of drugs for treating Gram-positive bacterial infections. Background Art

[0002] In recent years, the widespread use of antibiotics has led to a significant acceleration in the emergence and spread of drug-resistant bacteria, becoming a major problem in the field of public health. This phenomenon has made many common infections difficult to treat, posing a huge challenge to antibacterial treatment regimens in clinical practice. The spread of drug-resistant bacteria not only increases the morbidity of patients but also prolongs the hospital stay, resulting in a substantial increase in medical costs. More seriously, the rapid evolution and exacerbation of antibacterial drug resistance have severely weakened the efficacy of traditional antibacterial drugs, making the treatment of bacterial infection-related diseases more complex and difficult, and patients facing higher medical risks and longer recovery periods.

[0003] In a large number of cases of bacterial infections, more than 80% of the cases show that bacteria form biofilms, which seriously hinder the diffusion and penetration of antimicrobial drugs and become one of the important causes of chronic infections and antibiotic resistance. Biofilms are mainly composed of extracellular polymeric substances (EPS), which is a complex network structure containing components such as extracellular polysaccharides, proteins, and extracellular DNA. This structure not only provides a protective microenvironment for bacteria but also forms a barrier in terms of morphology, enabling bacteria to closely aggregate within the biofilm and jointly resist the invasion of external antibiotics. Under the protection of the biofilm, bacteria can evade the recognition of the immune system and the killing of antibacterial drugs, resulting in the ineffective control of infections caused by related pathogens and often leading to the failure of clinical anti-infection treatment. Therefore, it is of great practical significance to develop new antibacterial drugs that can disrupt biofilms and eliminate drug-resistant strain infections. Summary of the Invention

[0004] In view of the above technical problems, the present invention discloses the use of AZD-5991 in the preparation of drugs for treating Gram-positive bacterial infections. AZD-5991 shows antibacterial activity against clinically isolated Gram-positive bacteria including Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, etc. In addition, AZD-5991 has bactericidal activity against Staphylococcus aureus, can kill bacteria in mature biofilms, and can inhibit the formation of biofilms of Staphylococcus aureus and Enterococcus faecalis.

[0005] For this, the technical solution adopted by the present invention is as follows:

[0006] Use of AZD-5991 in the preparation of a medicament for treating Gram-positive bacterial infections. The AZD-5991 has a CAS number of 2143061-81-6, and AZD-5991 has the effect of inhibiting the growth and biofilm formation of Gram-positive bacteria.

[0007] Among them, the structural formula of the AZD-5991 is shown in formula (1):

[0008]

[0009] AZD-5991 is a highly selective MCL-1 inhibitor that can directly bind to the MCL-1 protein and block its interaction with pro-apoptotic proteins (such as BAK and BAX). AZD-5991 is overexpressed in a variety of hematological malignancies (such as multiple myeloma, acute myeloid leukemia) and certain solid tumors, and AZD-5991 may have therapeutic potential for these cancers. So far, no reports on the antibacterial activity of the AZD-5991 compound have been seen.

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

[0011] Through a large number of experiments, it is found that the AZD-5991 compound shows antibacterial activity against clinical isolates of Gram-positive bacteria including Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium. Moreover, AZD-5991 has bactericidal activity against Staphylococcus aureus, can also kill bacteria in mature biofilms, and significantly inhibits the formation of biofilms of Staphylococcus aureus and Enterococcus faecalis. AZD-5991 can inhibit the planktonic growth and biofilm formation of Staphylococcus aureus by disrupting the cell membrane and targeting FabI.

[0012] As a further improvement of the present invention, the concentration of the AZD-5991 in the treatment system is not less than 6.25 μM. Further, the concentration of the AZD-5991 compound in the treatment system is not less than 12.5 μM. Further, the concentration of the AZD-5991 compound in the treatment system is not less than 25 μM.

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

[0014] The present invention also discloses the use of AZD-5991 in the preparation of a coating for inhibiting Gram-positive bacteria, which is used on the surface of medical devices. The CAS number of AZD-5991 is 2143061-81-6, and the structural formula is shown in Formula (1); 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 invention, in the coating, the concentration of AZD-5991 is not less than 6.25 μM.

[0016] The present invention also discloses the use of AZD-5991 in the preparation of an anti-Gram-positive bacteria disinfectant. The CAS number of AZD-5991 is 2143061-81-6, and the structural formula is shown in Formula (1); this disinfectant has good antibacterial or bactericidal effects on a variety of Gram-positive bacteria including Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, etc. Further, in the anti-Gram-positive bacteria disinfectant, the concentration of AZD-5991 is not less than 6.25 μM.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The technical solution of the present invention discloses a new medical use of the AZD-5991 compound. The AZD-5991 compound shows antibacterial activity against clinical isolates such as Gram-positive bacteria including Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium. Moreover, it has bactericidal activity against Staphylococcus aureus, and can also kill bacteria in mature biofilms. AZD-5991 can inhibit the planktonic growth and biofilm formation of Staphylococcus aureus by disrupting the cell membrane and targeting FabI. It can be seen that the AZD-5991 compound has potential application value in clinical anti-bacterial infection treatment. Description of the Drawings

[0019] Figure 1 It is the growth curves of Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis after AZD-5991 in the examples of the present invention. In the figure, A and B are methicillin-resistant Staphylococcus aureus (MRSA) YUSA139 and YUSA145 respectively; C and D in the figure are the experimental results of clinical isolates EF16C30 and EF16C51 of Enterococcus faecalis respectively. MIC in the figure represents the minimum inhibitory concentration.

[0020] Figure 2This is the analysis of the bactericidal curve of AZD-5991 in the embodiments of the present invention against Gram-positive bacterium Staphylococcus aureus. In the figure, A shows the experimental results of the methicillin-resistant Staphylococcus aureus (MRSA) clinical strain YUSA145; B shows the methicillin-sensitive Staphylococcus aureus (MSSA) clinical strain CHS101.

[0021] Figure 3 This is the analysis of the inhibition of biofilm formation of Gram-positive bacterium Staphylococcus aureus and Enterococcus faecalis by AZD-5991 in the embodiments of the present invention. In the figure, A shows the quantitative analysis of the effect of AZD-5991 on the biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA) by crystal violet staining; B shows the quantitative analysis of the effect of AZD-5991 on the biofilm formation of methicillin-sensitive Staphylococcus aureus (MSSA) by crystal violet staining; C shows the quantitative analysis of the effect of AZD-5991 on the biofilm formation of Enterococcus faecalis (E.faecalis) by crystal violet staining. In the figure, MIC represents the minimum inhibitory concentration.

[0022] Figure 4 This is the effect of AZD-5991 on the cell membrane permeability of Staphylococcus aureus in the embodiments of the present invention. In the figure, A shows the quantitative analysis of the effect of AZD-5991 on the cell membrane potential of methicillin-sensitive Staphylococcus aureus (MSSA) SA113 by fluorescence intensity; B shows the quantitative analysis of the effect of AZD-5991 on the cell membrane permeability of methicillin-sensitive Staphylococcus aureus (MSSA) SA113 by fluorescence intensity.

[0023] Figure 5 This is the effect of adding bacterial membrane phospholipid component cardiolipin (CL) on the antibacterial activity of AZD-5991 in the embodiments of the present invention.

[0024] Figure 6 This is the cytotoxicity test results of AZD-5991 on human hepatoma cells HepG2, human hepatic stellate cells LX-2, human colon cancer cells HCT116, human lung alveolar basal epithelial cells A549 and human renal epithelial cell line 293T in the embodiments of the present invention.

[0025] Figure 7 This is the experiment on the effect of exogenous fatty acids on the antibacterial activity of AZD-5991 in the embodiments of the present invention. In the figure, A and B respectively show the effect of the addition of exogenous fatty acids linoleic acid (C18:2Δ9,12) and arachidonic acid (C20:4Δ5,8,11,14) on the MIC value of AZD-5991 against Staphylococcus aureus SA113; C shows the molecular docking results of predicting the binding pocket of AZD-5991 and FabI; D shows the binding kinetics results of analyzing the binding of AZD-5991 and FabI by BLI experiment. Detailed implementation manners

[0026] The following further elaborates on the preferred embodiments of the present invention.

[0027] Example 1

[0028] The 97 clinical strains of Gram-positive bacteria (including 20 strains of MRSA, 17 strains of MSSA, 21 strains of Staphylococcus epidermidis, 19 strains of Enterococcus faecium, and 20 strains of Enterococcus faecalis) used in this example were collected from different inpatients in the hospital. All clinical strains were identified by the Phoenix 100 automated microbial identification system, and after subculture, all strains were re-identified by matrix-assisted laser desorption ionization / time-of-flight mass spectrometry (MALDI-TOF-MS). The quality control strain Staphylococcus aureus SA113 (ATCC35556) was purchased from the ATCC strain bank.

[0029] In this example, a 96-well plate was used for high-throughput detection of the effect of AZD-5991 on the growth of Staphylococcus aureus and Enterococcus faecalis. The specific steps were as follows:

[0030] Take the overnight culture broth (Staphylococcus aureus SA113 and Enterococcus faecalis OG1RF) and adjust the turbidity to 0.5 McFarland (the bacterial amount is about 1.0 - 1.5×10 8 CFU / mL) with a turbidimeter cup. Dilute the bacterial broth 1:200 with CAMHB medium and add it to a 96-well plate, with 12 wells in each row and 200 μL in each well. Dilute the compounds in the custom compound library to 50 μM, and add 200 μL of CAMHB medium to the 12th well as a negative control. And use compounds such as PIM-447 (dihydrochloride), PF-06282999, 7ACC1, N-desmethylEnzalutamide, Glucagon receptor antagonists-3, IDO-IN-4, Zorifertinib, JAK3-IN-1, CY7-SE, JNJ-46778212, Yoda 1, SCR-1481B1, BPK-29 (hydrochloride), Cdc7-IN-1, AZD5904, etc. as control samples. Observe the results after culturing at 37°C for 24 hours. The compounds added to the wells where no bacterial growth can be seen with the naked eye are considered to have potential antibacterial activity.

[0031] Through experiments, it was found that the culture media of Staphylococcus aureus SA113 and Enterococcus faecalis OG1RF added with 50 μM AZD-5991 were both clear after 24 hours, and no bacterial growth was observed. The OD 600 was all less than 0.1; none of the other control sample compounds had significant antibacterial activity. It can be seen that AZD-5991 has potential antibacterial activity against Staphylococcus aureus and Enterococcus faecalis.

[0032] Example 2

[0033] In this example, the minimum inhibitory concentration (MIC) of AZD-5991 against 97 Gram-positive bacteria (including 20 strains of MRSA, 17 strains of MSSA, 21 strains of Staphylococcus epidermidis, 19 strains of Enterococcus faecium, and 20 strains of Enterococcus faecalis) was detected by the microbroth dilution method. The specific steps were as follows:

[0034] Take the overnight culture broth and adjust the turbidity to 0.5 McFarland (the bacterial amount is about 1.0 - 1.5×10 8 CFU / mL) using a turbidimeter. Dilute the bacterial solution 1:100 with CAMHB medium and add it to a 96-well plate, with 12 wells in each row. Set up a gradient of 9 concentration wells for the drug (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.7812, 0.3906 μM). Add 200 μL of the above-mentioned bacterial solution to the 11th well as a positive control, and add 200 μL of CAMHB medium to the 12th well as a negative control. Observe the results after culturing at 37°C for 18 hours, and calculate the MIC value based on the well with the drug concentration at which the bacterial solution precipitate cannot be seen with the naked eye.

[0035] In this example, the statistical results of the MIC values of AZD-5991 against Gram-positive bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium are shown in Table 1. It can be seen that AZD-5991 has relatively broad-spectrum antibacterial activity against various Gram-positive bacteria, and the MIC values are mainly distributed between 6.25 μM and 25 μM. Among them, the inhibitory effect on Staphylococcus aureus and Staphylococcus epidermidis is the best, and the MIC 50 is 6.25 μM.

[0036] Table 1 Distribution of MIC values of common Gram-positive bacteria against AZD-5991

[0037]

[0038] Note: MIC: Minimum inhibitory concentration; MSSA: Methicillin-sensitive Staphylococcus aureus; MRSA: Methicillin-resistant Staphylococcus aureus; S. epidermidis: Staphylococcus epidermidis; E. faecium: Enterococcus faecium; E. faecalis: Enterococcus faecalis; n is the number of tested strains. MIC 50 : The drug concentration required to inhibit the growth of 50% of the tested bacteria; MIC 90 : The drug concentration required to inhibit the growth of 90% of the tested bacteria.

[0039] Example 3

[0040] Experiment on the growth effect of AZD-5991 on Gram-positive and Gram-negative bacteria.

[0041] To verify whether AZD-5991 can inhibit the growth of bacteria, we treated two strains of Staphylococcus aureus (YUSA139 and YUSA145) and two strains of Enterococcus faecalis (EF16C30 and EF16C51) with different sub-inhibitory concentrations of AZD-5991 respectively, and measured the absorbance at 600 nm wavelength (OD 600 ) value at different time points. The specific steps are as follows:

[0042] Take the overnight culture broth, dilute it 1000 times with tryptic soy broth (TSB) medium, add it to a 96-well plate, add different concentrations (1 / 16×, 1 / 8×, 1 / 4×, 1 / 2× and 1×) of AZD-5991, and then place it in a full-automatic growth curve analyzer. Measure the absorbance at 600 nm wavelength (OD 600 ) absorbance every 1 hour to detect the content of planktonic bacteria in the culture supernatant. The incubation temperature is 37 °C, and the growth curve is plotted.

[0043] The growth curve analysis is as Figure 1 shown. It can be seen that at the 1 / 2×MIC concentration, AZD-5991 significantly inhibits the growth of planktonic Staphylococcus aureus, and when the concentration reaches 1×MIC, the growth of planktonic bacteria is completely inhibited. At the 1 / 4×MIC concentration, AZD-5991 can significantly inhibit the growth of planktonic Enterococcus faecalis, and when the concentration reaches 1×MIC, the growth of planktonic bacteria is basically inhibited. These results preliminarily indicate that AZD-5991 has the potential to be used as an anti-Gram-positive bacterial infection drug.

[0044] Example 4

[0045] Experiment on the effects of time and dose of AZD-5991 on the bactericidal activity against Gram-positive bacteria.

[0046] In this example, the time- and dose-dependent effects of AZD-5991 on the antibacterial activity against Gram-positive bacteria were studied, and the activity was compared with that of the clinical antibiotic vancomycin. The specific steps include:

[0047] Dilute the bacterial suspensions of the logarithmic phase (OD 600 = 0.5) of the MSSA clinical strain CHS101 and the MRSA clinical strain YUSA145 100 times, and incubate them with 1×, 2×, 4×, 8×MIC of AZD-5991 and 4×MIC of vancomycin in a shaker at 200 rpm. Subsequently, samples were collected at 0, 2, 6, and 24 hours respectively, serially diluted with sterile normal saline, and spread on TSB plates and incubated at 37 °C. Colony counting was performed after 24 hours. Colony counting is expressed as CFU / mL.

[0048] The obtained bactericidal curve analysis is asFigure 2 As shown, in the YUSA145 strain, AZD-5991 at a concentration of 1×MIC exhibited similar bactericidal activity to vancomycin at a concentration of 4×MIC; while in the CHS101 strain, AZD-5991 at a concentration of 4×MIC showed similar bactericidal effects to vancomycin at a concentration of 4×MIC. In addition, at a concentration of 8×MIC, AZD-5991 showed a potent clearance effect on the YUSA145 strain after 4 hours of drug exposure, indicating that AZD-5991 has rapid and efficient bactericidal activity against planktonic bacteria.

[0049] Example 5

[0050] In this example, the crystal violet method was used to study the inhibitory biofilm formation activity of AZD-5991 against Staphylococcus aureus and Enterococcus faecalis strains. The specific steps were as follows:

[0051] The crystal violet staining method was used to detect the change in biofilm amount, repeated 3 times, with 3 replicates in each time, and the average value was taken as the final detection result. The operation steps are briefly described as follows: The overnight bacterial solution was diluted 1000-fold with TSBG medium containing 2% glucose in a 96-well plate, with 3 replicates in each group. Different sub-inhibitory concentrations of AZD-5991 were added, and the blank medium without bacterial solution 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. Gently aspirate and discard the culture medium, rinse 3 times with sterile PBS, and air dry at room temperature. Fix with methanol for 15 minutes, stain with 1% crystal violet solution for 15 minutes, rinse 3 times with sterile water until the control wells are colorless, and air dry at room temperature. Add 200 μL of absolute ethanol to each well to dissolve, shake for 1 minute, and measure the absorbance at 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader.

[0052] The results of crystal violet staining are as Figure 3 shown. It can be seen that different sub-inhibitory concentrations of AZD-5991 have a significant inhibitory effect on the formation of biofilms of Enterococcus faecalis and Staphylococcus aureus; when AZD-5991 is 1 / 2×MIC, for all detected Staphylococcus aureus strains, whether MSSA or MRSA, the biomass of the formed biofilm is significantly reduced, and the formation of its biofilm is inhibited by more than 50%. When AZD-5991 is 1 / 2×MIC, the biomass of the biofilm of Enterococcus faecalis strains is reduced, and when AZD-5991 is 1×MIC, the biomass of the biofilm of Enterococcus faecalis strains is greatly inhibited. The above results indicate that AZD-5991 can significantly inhibit the formation of biofilms of Staphylococcus aureus and Enterococcus faecalis.

[0053] Example 6

[0054] This example is an experimental study on the cell membrane potential and permeability of SA113 of Staphylococcus aureus by AZD-5991. The specific steps are as follows:

[0055] Membrane potential experiment: The Gram-positive bacterium SA113 cells in the logarithmic phase were adjusted to an OD 600 = 0.05 and incubated with 2 μM SYTOX Green in 5 mM HEPES buffer (pH = 7.2) in the dark. The suspension was treated with different concentrations of AZD-5991 (final concentrations of 1×, 2×, and 4× MIC), with 0.1% DMSO and 0.1% Triton as controls, and the fluorescence intensity was continuously monitored (excitation wavelength: 504 nm, emission wavelength: 523 nm). The above-mentioned suspended logarithmic cells and 2 μM DiBAC4(3) were suspended for 5 minutes. After treatment with different concentrations of AZD-5991, the fluorescence intensity was monitored at an excitation wavelength of 622 nm and an emission wavelength of 670 nm.

[0056] Membrane permeability experiment: The Gram-positive bacterium SA113 cells in the logarithmic phase were adjusted to an OD 600 = 0.05 and incubated with propidium iodide (PI) fluorescent dye in the dark. Then the suspension was treated with different concentrations of AZD-5991 (final concentrations of 1×, 2×, and 4× MIC), with 0.1% DMSO and 0.1% Triton as controls, and the fluorescence value of propidium iodide (PI) staining was monitored with an enzyme-linked immunosorbent assay (ELISA) reader after 1 hour.

[0057] The monitoring results of the fluorescence intensity are as Figure 4 shown. It can be seen that after treatment with AZD-5991, Staphylococcus aureus SA113 depolarized the cell membrane at 1× MIC or 2× MIC concentrations, similar to the effect of 0.1% Triton X-100. In addition, under AZD-5991 exposure, the fluorescence intensity of PI staining in SA113 cells at 4× MIC concentration increased by 1.2 times compared with the control group, indicating a change in cell membrane permeability and that AZD-5991 has a damaging effect on the cell membrane of Staphylococcus aureus.

[0058] Example 7

[0059] In this example, the antibacterial activity of AZD-5991 acting on the phospholipid components of the bacterial cell membrane was studied. The specific steps include:

[0060] In this example, in order to analyze whether the antibacterial activity of AZD-5991 is related to the cell membrane, the checkerboard microbroth dilution method was used to detect the effect of cardiolipin (CL), a phospholipid component of the bacterial cell membrane, on the minimum inhibitory concentration (MIC) of AZD-5991. The specific steps are as follows:

[0061] The overnight culture broth of Staphylococcus aureus CHS101 was taken and the turbidity was adjusted to 0.5 McFarland (the bacterial amount was about 1.0 - 1.5×10 8CFU / mL). The bacterial solution was diluted 1:100 with CAMHB medium and added to a 96-well plate, with 12 wells in each row. AZD-5991 was set with 8 gradient wells (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 μM). 200 μL of the above bacterial solution was added to the 9th well as a positive control, and 200 μL of CAMHB medium was added to the 10th well as a negative control; 11 gradient wells (128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 μg / mL) were set for the cell membrane phospholipid components. The MIC values of each antibacterial drug were determined according to the CLSI guidelines for the culture conditions and time. After culturing at 37 °C for 18 hours, the results were observed, and the drug concentration well where the bacterial solution precipitation could not be seen with the naked eye was calculated as the MIC value.

[0062] The results obtained were plotted with the concentration of different phospholipids on the abscissa and the fold change in the MIC concentration of AZD-5991 on the ordinate. The results were as Figure 5 shown. Adding cardiolipin (CL) exogenously to the medium reduced the antibacterial activity of AZD-5991 in a dose-dependent manner, indicating that AZD-5991 exerted its antibacterial activity by acting on the bacterial cell membrane.

[0063] Example 8

[0064] This example was an experiment on the cytotoxicity of AZD-5991 on human hepatocellular carcinoma cells HepG2, human hepatic stellate cells LX-2, and human renal epithelial cell line 293T. The specific steps were as follows:

[0065] 100 μL of cell suspensions of human hepatocellular carcinoma cells HepG2, human hepatic stellate cells LX-2, human colon cancer cells HCT116, human alveolar basal epithelial cells A549 of lung cancer, and human renal epithelial cell line 293T were prepared in a 96-well plate. The culture plate was pre-cultured in an incubator for 24 hours (37 °C, 5% CO2). 10 μL of different concentrations of AZD-5991 was added to the culture plate, with 8 gradient wells set (100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 μM). The culture plate was incubated in the incubator for 24 hours, and 10 μL of CCK-8 solution was added to each well. The culture plate was incubated in the incubator for 1 - 4 hours. The absorbance at 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader. Then the cell viability was calculated from the absorbance.

[0066] CCK-8 kit is a rapid and highly sensitive detection kit based on WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt), which is widely used for the detection of cell proliferation and cytotoxicity. Its working principle is as follows: in the presence of an electron coupling reagent, it can be reduced by dehydrogenases in mitochondria to form a highly water-soluble orange-yellow formazan product. The intensity of the color is directly proportional to cell proliferation and inversely proportional to cytotoxicity. The OD450 value is measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader to indirectly reflect cell viability. The results of the cytotoxicity of AZD-5991 against HepG2, LX-2, HCT116, A549, and 293T cells are as Figure 6 shown. It can be seen that AZD-5991 has relatively low cytotoxicity, higher than the MIC values against most Gram-positive clinical strains tested, indicating its potential for clinical anti-infective applications.

[0067] Example 9

[0068] This example is an experiment on detecting gene mutations induced by AZD-5991-Staphylococcus aureus YUSA145 using whole-genome sequencing. The specific steps are as follows:

[0069] Through 40 consecutive passages in vitro, Staphylococcus aureus YUSA145 was induced to develop resistance to AZD-5991 under the pressure of AZD-5991. Finally, the AZD-5991-resistant strain YUSA145N40 was screened out, and its minimum inhibitory concentration was determined. It was found that its MIC increased by 8-fold compared to the original strain, reaching 100 μM. Subsequently, the genetic mutations of this resistant strain were detected by whole-genome sequencing to study its resistance mechanism. The results of the whole-genome sequencing are shown in Table 2. It can be seen that three non-synonymous mutations occurred in the AZD-5991-resistant strain YUSA145N40, located on three functional genes, including the gene encoding the fatty acid efflux pump transcriptional regulator FarR, the gene encoding the helix-turn-helix structural protein, and the gene encoding threonylcarbamoyl-AMP synthase. FarR plays an important role in long-chain fatty acid metabolism. Based on these mutations, it is speculated that the exposure to AZD-5991 may affect cell membrane synthesis and fatty acid metabolic pathways.

[0070] Table 2 Results of whole-genome sequencing analysis of gene mutations in YUSA145N40

[0071]

[0072] Note: nonsyn: non-synonymous mutation; stop_mutation: termination mutation; NC mutations: nucleotide mutation sites; AA mutations: amino acid mutation sites.

[0073] Example 10

[0074] This example is an experiment to study the effect of AZD-5991 on the fatty acid metabolic pathway. The specific steps are as follows:

[0075] Take the overnight culture broth of Staphylococcus aureus CHS101 and adjust the turbidity to 0.5 McFarland (the bacterial amount is about 1.0 - 1.5×10 8 CFU / mL) using a turbidimeter. Dilute the bacterial solution 1:100 with CAMHB medium and add it to a 96-well plate, with 12 wells in each row. Set 8 gradient wells for AZD-5991 (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 μM). Add 200 μL of the above-mentioned bacterial solution to the 9th well as the positive control, and add 200 μL of CAMHB medium to the 10th well as the negative control; set 7 gradient wells for fatty acids (500, 250, 125, 62.5, 31.25, 15.6, 7.8 μM). The culture conditions and time for measuring the MIC value of each antibacterial drug are carried out according to the CLSI guidelines. Observe the results after culturing at 37°C for 18 hours, and calculate the MIC value based on the drug concentration well where the bacterial solution precipitate cannot be seen with the naked eye. The monitoring results of fluorescence intensity are as Figure 7 A, Figure 7 shown in B. It was found that linoleic acid and arachidonic acid could significantly increase the MIC value of AZD-5991 and reduce its antibacterial activity against Staphylococcus aureus in a concentration-dependent manner.

[0076] Then, a molecular docking prediction experiment was carried out. Use AlphaFoldDB to generate the molecular structure PDB file of FabI (Uniprot ID: Q2FVQ3), and then use the Protein Preparation Wizard function of the software to perform hydrogenation, supplement missing amino acid residues, optimize the structure and water molecules, obtain the 3D molecular structure file of AZD-5991 from PubChem, and then establish the binding framework between FabI protein and AZD-5991 through molecular docking. Finally, select the best binding site of FabI protein using structure-based cavity detection and evaluate the best binding position of AZD-5991 at this site using AutoDock Vina. The results are as Figure 7 shown in C. The molecular docking results show that AZD-5991 can bind to FabI with a good conformation, and the binding energy is -8.3 kcal / mol, indicating a strong binding force.

[0077] Finally, BLI kinetic experiments were used to confirm the binding of AZD-5991 to FabI. Biotinylated FabI was immobilized on a streptavidin sensor and treated with kinetic buffer (PBS, 0.05% bovine serum albumin, 0.01% Tween 20). Subsequently, the sensor was exposed to different concentrations of AZD-5991, and protein-free buffer treatment was performed using a repeated background control group sensor to correct for non-specific binding and signal variation. The experiment was carried out in a 96-well plate with the temperature set at 30 °C and the volume per well at 300 μL. After the experiment, data analysis was performed using Gatorprime software, and the double reference subtraction method was used to accurately determine the binding kinetics. The results are as Figure 7 shown in D, indicating a direct binding between AZD-5991 and FabI. In summary, the research results suggest that AZD-5991 may exert its antibacterial effect by affecting the fatty acid metabolic pathway.

[0078] All of the above experiments were processed for data and plotted images using GraphPad Prism 8.0 software. P ≤ 0.05 was considered to be statistically significant.

[0079] As can be seen from the experimental results of the above examples, the AZD-5991 compound exhibits good antibacterial activity against common clinical Gram-positive bacteria. AZD-5991 has a significant bactericidal effect on Staphylococcus aureus, and even at 8×MIC concentration, the bacterial count is reduced to the lower limit of detection within 4 hours. In addition, AZD-5991 exhibits good anti-biofilm activity and can significantly inhibit the formation of biofilms by clinical strains of Staphylococcus aureus and Enterococcus faecalis. AZD-5991 has low toxicity to HepG2, LX-2, and 293T cells at the MIC dose. The addition of exogenous fatty acids linoleic acid (C18:2Δ9,12) and arachidonic acid (C20:4Δ5,8,11,14) significantly enhances the antibacterial activity of AZD-5991. Finally, biolayer interferometry analysis supports the direct interaction between AZD-5991 and FabI, which is an essential protein closely related to bacterial growth and fatty acid metabolism. This study shows that AZD-5991 can inhibit the planktonic growth and biofilm formation of Staphylococcus aureus by disrupting the cell membrane and targeting FabI. These results suggest the possibility of the AZD-5991 compound for treating clinical bacterial infectious diseases. Moreover, the MIC of AZD-5991 is comparable to that of commonly used clinical antibiotics, making it suitable for clinical use.

[0080] The above content is a further detailed description of the present invention in combination with specific preferred embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, and all should be regarded as belonging to the protection scope of the present invention.

Claims

1. The use of AZD-5991 in the preparation of a drug for anti-Gram-positive bacterial infection, characterized in that: The AZD-5991 has a CAS number of 2143061-81-6; the Gram-positive bacteria is at least one of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium.

2. The use of AZD-5991 according to claim 1 for preparing a drug for anti-Gram-positive bacterial infection, characterized in that: The concentration of AZD-5991 in the treatment system is not less than 6.25 μM.

3. The use of AZD-5991 according to claim 1 for preparing a drug for anti-Gram-positive bacterial infection, characterized in that: The medicine is in the form of injection, tablet, pill, capsule, suspension, granule, spray or emulsion.

4. The use of AZD-5991 in the preparation of a coating for inhibiting Gram-positive bacteria, characterized in that: The coating is used for the surface of medical devices, the CAS number of the AZD-5991 is 2143061-81-6, and the Gram-positive bacteria is at least one of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium.

5. The use of AZD-5991 according to claim 4 for preparing a coating for inhibiting Gram-positive bacteria, characterized in that: In the coating, the concentration of AZD-5991 is not less than 6.25 μM.

6. The use of AZD-5991 for preparing an anti-Gram-positive bacteria disinfectant, characterized in that: The CAS number of the AZD-5991 is 2143061-81-6, and the Gram-positive bacteria is at least one of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium.

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