Application of compound 5928-0054 in preparation of anti-staphylococcus aureus hemolysin medicine
By neutralizing the toxicity of Staphylococcus aureus hemolysin by compound 5928-0054, the problem that existing antibiotics cannot alleviate inflammation and drug resistance is solved, and effective protection and treatment of Staphylococcus aureus infection is achieved.
Patent Information
- Application Number
- CN202411702265.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2023-11-29
- Filing Date
- 2024-11-26
- Publication Date
- 2025-02-14
AI Technical Summary
Existing antibiotics cannot effectively alleviate the impact of inflammation caused by Staphylococcus aureus infection on the body, and the problem of bacteria resistance is becoming increasingly serious.
Compound 5928-0054 is able to neutralize the toxicity of Staphylococcus aureus hemolysin, inhibiting its hemolytic activity by hydrogen bonding with the hemolysin protein, thereby providing protection against Staphylococcus aureus infection.
Compound 5928-0054 significantly inhibited the hemolytic activity of Staphylococcus aureus, reduced the damage to the body by inflammation, and reduced the bacterial mutation rate, providing new drugs to prevent and treat Staphylococcus aureus infection.
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Figure CN119424415A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of microbial infectious diseases and medical technology, and specifically to application of compound 5928-0054 in the preparation of drugs against Staphylococcus aureus hemolysin. Background Art
[0002] Staphylococcus aureus, as one of the multidrug-resistant (MDR) and extensively drug-resistant (XDR) bacteria, is an asymptomatic colonizer of 30% of humans and the most dangerous of all staphylococci. Currently, methicillin-resistant Staphylococcus aureus (MRSA) causes 10 times more infections than all multidrug-resistant (MDR) Gram-negative pathogens combined. Recently, MRSA was listed by the World Health Organization (WHO) as one of the twelve priority pathogens threatening human health. According to reports, the highest incidence of MRSA (>50%) is in North America, South America, Asia and Malta. The incidence in China, Australia, Africa and several European countries is at a moderate level (25-50%), while the incidence in most European countries is low, causing huge economic losses to public health and the aquaculture industry. α-hemolysin is the most famous toxin of Staphylococcus aureus, which destroys the cell membrane structure and causes apoptosis of human monocytes, T cells and B cells. Although commonly used antibiotics can kill Staphylococcus aureus, they cannot alleviate the effects of inflammation caused by Staphylococcus aureus on the body. Summary of the invention
[0003] The purpose of the present invention is to provide a new application of compound 5928-0054, that is, to provide the application of compound 5928-0054 in the preparation of a drug against Staphylococcus aureus hemolysin. The structure of the compound 5928-0054 is shown below:
[0004]
[0005] The purpose of the present invention is achieved through the following technical solutions:
[0006] The present invention found that compound 5928-0054 can neutralize the toxicity of Staphylococcus aureus hemolysin and has a protective effect on mice infected with Staphylococcus aureus. Based on this, the present invention provides the following applications:
[0007] Use of compound 5928-0054 or a pharmaceutically acceptable salt thereof in preparing a drug for resisting Staphylococcus aureus hemolysin.
[0008] Use of compound 5928-0054 or a pharmaceutically acceptable salt thereof in preparing a drug for preventing or treating Staphylococcus aureus infection.
[0009] Use of compound 5928-0054 or a pharmaceutically acceptable salt thereof in preparing a drug for resisting bacterial hemolysin.
[0010] Use of compound 5928-0054 or a pharmaceutically acceptable salt thereof in preparing a drug for preventing or treating hemolysin-producing bacterial infection.
[0011] The present invention has the following advantages and beneficial effects:
[0012] (1) The present invention discovered the new medicinal value of 5928-0054 and provided a new preventive and therapeutic drug for Staphylococcus aureus infection.
[0013] (2) 5928-0054 can neutralize hemolysin, the main virulence factor of Staphylococcus aureus, and reduce the damage of inflammation to the body.
[0014] (3) 5928-0054 works by neutralizing bacterial virulence factors, thereby reducing the bacterial mutation rate. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 This is a statistical chart of the hemolytic toxicity results of 5928-0054 on red blood cells.
[0016] Figure 2 This is a statistical chart of the lysis results of red blood cells by the culture supernatant of Staphylococcus aureus USA300.
[0017] Figure 3 This is a statistical graph showing the results of 5928-0054 inhibiting the hemolytic activity of the culture supernatant of Staphylococcus aureus USA300.
[0018] Figure 4 This is a statistical graph showing the results of 5928-0054 inhibiting the hemolytic activity of Staphylococcus aureus USA300 hemolysin protein.
[0019] Figure 5 This is a statistical graph showing the results of 5928-0054 inhibiting the hemolytic activity of Streptococcus suis SC19 hemolysin protein.
[0020] Figure 6 This is a molecular docking model diagram of Staphylococcus aureus α-hemolysin and 5928-0054, A: docking conformation of 6U49 and 5928-0054; B: 5928-0054 is bound to the active domain of 6U49 protein through three hydrogen bonds (dashed lines); C: the highest affinity conformation between 6U49 and 5928-0054.
[0021] Figure 7 It is a heat molar ratio curve of 5928-0054 and Staphylococcus aureus hemolysin protein.
[0022] Figure 8 It is the kinetic reaction curve of 5928-0054 and Staphylococcus aureus hemolysin protein.
[0023] Fig. 9 It is a graph of the survival curves of mice 5928-0054 treated and untreated infected with Staphylococcus aureus USA300.
[0024] Fig.10 This is a graph showing the detection results of IL-6 and TNF-α in mouse serum.
[0025] Fig.11 This is a graph showing the results of blood biochemical level testing in mice.
[0026] Fig.12 This is a graph showing the results of bacterial load detection in the lungs, spleen, kidneys, and liver of mice. DETAILED DESCRIPTION
[0027] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
[0028] The Staphylococcus aureus USA300 used in the following examples is a standard strain of methicillin-resistant Staphylococcus aureus (MRSA); the statistical analysis of the results of the examples was performed using a two-tailed unpaired t-test, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001.
[0029] Example 1
[0030] (1) In a 96-well plate, 50 μL of 2% sheep red blood cells suspended in PBS were added to 50 μL of 5928-0054 serially diluted in PBS and incubated at 37°C for 1 hour to make the final concentrations of 5928-0054 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL and 128 μg / mL, and the wells in which the red blood cells were added with an equal amount of PBS were used as negative controls, and 2.5% TritonX-100 was used as a positive control. The plate was then centrifuged at 500 r / min for 5 min, and 50 μL of supernatant from each well of the assay plate was transferred to a fresh 96-well plate. Hemolysis was confirmed by measuring the absorbance at 543 nm.
[0031] Results Figure 1 , 5928-0054 itself cannot cause red blood cell hemolysis.
[0032] (2) Staphylococcus aureus USA300 was cultured at 37°C for 12 h, centrifuged at 4°C and 5000 r / min for 10 min, and the supernatant was collected. Different volumes of culture supernatant were added to phosphate buffered saline (PBS, pH=7.4) containing 2% sheep red blood cells (total volume 1 mL) and incubated at 37°C for 30 min. Finally, centrifuged at 500 r / min for 5 min at 4°C. Subsequently, 200 μL of supernatant was collected and its OD value was measured at 543 nm. At the same time, a PBS solution containing 2% sheep red blood cells was treated with 2.5% TritonX-100 and set as a 100% positive control. The hemolytic activity of the culture supernatant of Staphylococcus aureus USA300 was evaluated by the ratio of the OD543 value of each sample to the positive control. The results are shown in Figure 2 , 125 μL of the culture supernatant of Staphylococcus aureus USA300 can lyse about 90% of the red blood cells.
[0033] (3) Staphylococcus aureus USA300 was cultured at 37°C for 12h, centrifuged at 4°C and 5000r / min for 10min, and the supernatant was collected. 125μL of the supernatant containing different concentrations of 5928-0054 was incubated at 37°C for 30min. Then 875μL of PBS buffer solution containing 2% sheep red blood cells was added and incubated at 37°C for 30min. Finally, centrifuged at 1000r / min for 5min at 4°C, 200μL of the supernatant was collected, and the value at OD543nm was measured. At the same time, the solution containing 2% sheep red blood cells was treated with 2.5% TritonX-100 as a positive control. The effect of 5928-0054 on the hemolytic activity of the culture supernatant of Staphylococcus aureus USA300 was evaluated by the ratio of the OD543 value of each sample to the positive control.
[0034] Results Figure 3 , 5928-0054 significantly inhibited the hemolytic activity of the supernatant of Staphylococcus aureus USA300 in a concentration-dependent manner.
[0035] (4) The purified Staphylococcus aureus USA300 hemolysin protein (100 ng / mL) was co-incubated with different concentrations of 5928-0054, and the anti-Staphylococcus aureus hemolysin protein effect of 5928-0054 was evaluated according to the method in (3).
[0036] Results Figure 4 , consistent with the results in (3), 5928-0054 inhibited the hemolytic activity of Staphylococcus aureus USA300 hemolysin protein.
[0037] (5) The purified Streptococcus suis SC19 hemolysin protein SLY (100 ng / mL) was co-incubated with different concentrations of 5928-0054, and the anti-Streptococcus suis SC19 hemolysin protein SLY effect of 5928-0054 was evaluated according to the method in (3).
[0038] Results Figure 5 , indicating that 5928-0054 can also inhibit the hemolytic activity of swine streptococcal hemolysin protein SLY.
[0039] Example 2
[0040] (1) The 3D structure file of compound 5928-0054 was obtained from the Pubchem small molecule database (https: / / pubchem.ncbi.nlm.nih.gov / ), and the α-hemolysin protein structure of Staphylococcus aureus was obtained from the RCSB database (https: / / www.rcsb.org / ). The hemolysin protein (PDB ID: 6U49) was obtained by X-ray diffraction. The 3D structure of the hemolysin heptamer. The molecular docking process was completed by MOE 2019 software to predict the interaction conformation between compound 5928-0054 and the hemolysin protein. The hemolysin protein was completed and optimized using the QuickPrep module. Since the 6U49 structure contained a specific ligand, all ligands, water molecules, and ADP were removed. Compound 5928-0054 was prepared for ligands by assigning charges, generating conformers, and optimizing conformations. The binding pocket of the hemolysin protein was found using the Site finder module, and virtual particles were generated in the binding pocket.
[0041] The docking program uses Amber14: EHT force field to calculate the charges of all protein and ligand atoms. All ligand conformations were docked into the docking pocket occupied by the virtual particle. The triangle matcher method was used to position the ligand in the binding pocket, 15 docking simulations were performed, and the complex conformations were scored by the London dG function. The highest scoring conformation between compound 5928-0054 and 6U49 is shown in Figure 2. Figure 6 A, 5928-0054 and 6U49 formed three groups of interactions, all of which were hydrogen bonds. In the hemolysin heptamer, 5928-0054 formed hydrogen bonds with ASN 172, LYS154 of the A chain and LYS215 of the B chain of 6U49 ( Figure 6 B), the energy between the O atom of 5928-0054 and LYS154 of chain A is the highest, which is -7.8 kcal / mol, as shown in the schematic diagram Figure 6 C.
[0042] (2) Isothermal calorimetric titration (ITC) was used to determine the interaction between Staphylococcus aureus hemolysin protein and 5928-0054 in vitro. The purified hemolysin protein and 5928-0054 were dissolved in PBS at pH = 7.4 to a final concentration of 0.02 mmol / L and 0.2 mmol / L, respectively. The instrument was balanced to 25°C, and 5928-0054 (total volume of 50 μL) was injected into a sample pool of 300 μL purified hemolysin protein, 2.5 μL each time, with a reaction interval of 200 s. The heat molar ratio curve of 5928-0054 and hemolysin protein during the reaction process can be seen in Figure 7 The kinetic reaction curve of 5928-0054 and hemolysin protein was fitted by NanoAnalyzer software and the equilibrium dissociation constant (Kd) was calculated. The results are shown in Figure 8 , calculated Kd = 2.81 × 10 -7 mol / L, indicating that 5928-0054 has good binding affinity with Staphylococcus aureus hemolysin.
[0043] It can be seen from this example that 5928-0054 can also bind to other bacterial hemolysins with a structure similar to that of the Staphylococcus aureus hemolysin protein to inhibit their hemolytic activity.
[0044] Example 3
[0045] The mice used in the following experiments were seven-week-old female BALB / c mice.
[0046] (1) Survival rate experiment
[0047] Mice were randomly divided into two groups: the 5928-0054 treatment group and the untreated group, with 10 mice in each group. The overnight cultured Staphylococcus aureus USA300 was transferred into LB medium at a volume ratio of 1:100 and cultured at 37°C until OD600 = about 0.5. The cells were collected after centrifugation at 4°C and 4000r / min for 5 minutes, and resuspended and mixed with PBS for later use. In the survival rate experiment, each mouse was injected with 2.5×10 8 The number of CFU bacteria. Two hours after infection, mice were intraperitoneally injected with 5928-0054. The dosage was 5 mg / kg, once every 12 hours, and the treatment lasted for three days. The untreated group was injected with the same volume of PBS. The mouse survival curve constructed based on the experimental results is shown in Figure 2. Fig. 9 As shown, the survival rate of mice in the 5928-0054 treated group reached about 70% compared with the untreated group.
[0048] (2) Detection of inflammatory factors, blood biochemistry and tissue bacterial load
[0049] The mice were randomly divided into an untreated group, a 5928-0054-treated group, and a blank control group, with 5 mice in each group. The intraperitoneal injection concentration of the untreated group and the 5928-0054-treated group was 5×10 7 CFU of USA300, and the blank control group was injected with the same volume of PBS. Two hours after infection, the mice in the 5928-0054 treatment group were intraperitoneally injected with 5928-0054 at a dose of 5 mg / kg; the untreated group and the blank control group were injected with the same volume of PBS. Twelve hours after the injection, the mice were killed and blood and tissues were collected. The effects of 5928-0054 on the blood biochemical (ALT, AST, CK) levels of mice infected with USA300 were analyzed using a fully automatic blood biochemical analyzer. In addition, the lungs, spleen, kidneys and livers were ground, diluted, and inoculated onto LA plates, cultured overnight at 37°C, and the bacteria were counted. A sensitive electrochemiluminescence platform (Quickplex, MSD) was used to detect inflammatory cytokines.
[0050] The results are as follows:
[0051] like Fig.10 As shown, 5928-0054 could significantly inhibit the production of IL-6 and TNF-α in infected mice.
[0052] like Fig.11 As shown, 5928-0054 treatment of mice significantly reduced blood biochemical levels.
[0053] like Fig.12 As shown, 5928-0054 was able to significantly reduce the tissue bacterial load in the lungs, spleen, kidneys and liver of infected mice.
[0054] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.
Claims
1. Use of compound 5928-0054 in the preparation of a drug against Staphylococcus aureus hemolysin, characterized in that: The structure of the compound 5928-0054 is shown below:
2. Use of a pharmaceutically acceptable salt of compound 5928-0054 in the preparation of a drug against Staphylococcus aureus hemolysin, characterized in that: The structure of the compound 5928-0054 is shown below:
3. Use of compound 5928-0054 in the preparation of a medicament for preventing or treating Staphylococcus aureus infection, characterized in that: The structure of the compound 5928-0054 is shown below:
4. Use of a pharmaceutically acceptable salt of compound 5928-0054 in the preparation of a medicament for preventing or treating Staphylococcus aureus infection, characterized in that: The structure of the compound 5928-0054 is shown below:
5. Use of compound 5928-0054 in the preparation of a drug for antibacterial hemolysin, characterized in that: The structure of the compound 5928-0054 is shown below:
6. Use of a pharmaceutically acceptable salt of compound 5928-0054 in the preparation of a drug for antibacterial hemolysin, characterized in that: The structure of the compound 5928-0054 is shown below:
7. Use of compound 5928-0054 in the preparation of a medicament for preventing or treating hemolysin-producing bacterial infection, characterized in that: The structure of the compound 5928-0054 is shown below:
8. Use of a pharmaceutically acceptable salt of compound 5928-0054 in the preparation of a medicament for preventing or treating hemolysin-producing bacterial infection, characterized in that: The structure of the compound 5928-0054 is shown below: