Use of compound 5928-0054 in preparation of drug against staphylococcus aureus hemolysin
Compound 5928-0054 neutralizes the toxicity of Staphylococcus aureus hemolysin by binding with hydrogen bonds, thus solving the problem that existing antibiotics cannot alleviate inflammation. This achieves effective prevention and treatment of Staphylococcus aureus infection, improves the survival rate of infected mice, and reduces the bacterial load.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- SHANGHAI TOPSCIENCE CO LTD
- Filing Date
- 2025-10-22
- Publication Date
- 2026-06-04
AI Technical Summary
Existing antibiotics are ineffective in alleviating the effects of Staphylococcus aureus infection on the body, and the multidrug resistance and extensive drug resistance of Staphylococcus aureus make the infection difficult to control.
Compound 5928-0054 can neutralize the toxicity of Staphylococcus aureus hemolysin by binding to hemolysin protein via hydrogen bonds, thereby inhibiting its hemolytic activity and exhibiting a protective effect against Staphylococcus aureus infection in vivo.
Compound 5928-0054 significantly reduced the damage of inflammation to the body, decreased the bacterial mutation rate, improved the survival rate of infected mice, and reduced the bacterial load and inflammatory factor production at the infection site.
Smart Images
Figure CN2025129422_04062026_PF_FP_ABST
Abstract
Description
Application of compound 5928-0054 in the preparation of drugs against Staphylococcus aureus hemolysin Technical Field
[0001] This invention relates to the fields of microbial infectious diseases and pharmaceutical technology, specifically the application of compound 5928-0054 in the preparation of a drug for treating Staphylococcus aureus hemolysin. Background Technology
[0002] Staphylococcus aureus, one of the most multidrug-resistant (MDR) and extensively drug-resistant (XDR) bacteria, accounts for 30% of asymptomatic colonizations in humans and is the most dangerous of all staphylococci. Currently, methicillin-resistant Staphylococcus aureus (MRSA) infections are 10 times more numerous than infections caused by all 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. Reportedly, North America, South America, Asia, and Malta have the highest MRSA incidence rates (>50%). China, Australia, Africa, and several European countries have moderate incidence rates (25-50%), while most European countries have lower rates, resulting in significant economic losses to public health and the livestock industry. Alpha-hemolysin is the most well-known toxin of Staphylococcus aureus, which disrupts cell membrane structure and induces apoptosis in human monocytes, T cells, and B cells. While commonly used antibiotics can kill Staphylococcus aureus, they cannot alleviate the inflammatory effects caused by Staphylococcus aureus on the body. Summary of the Invention
[0003] The object of this invention is to provide a new application for compound 5928-0054, specifically, its use in the preparation of a drug for treating Staphylococcus aureus hemolysin. The structure of compound 5928-0054 is shown below:
[0004] The objective of this invention is achieved through the following technical solution:
[0005] This invention discovered that compound 5928-0054 can neutralize the toxicity of Staphylococcus aureus hemolysin and has a protective effect against Staphylococcus aureus infection in mice. Based on this, this invention provides the following applications:
[0006] The use of compound 5928-0054 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the use of an anti-staphylococcal hemolysin.
[0007] The use of compound 5928-0054 or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention or treatment of Staphylococcus aureus infections.
[0008] Use of compound 5928-0054 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for antibacterial hemolysin.
[0009] The use of compound 5928-0054 or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention or treatment of hemolysin-producing bacterial infections.
[0010] The present invention has the following advantages and beneficial effects:
[0011] (1) This invention has discovered new medicinal value for 5928-0054 and provides new drugs for the prevention and treatment of Staphylococcus aureus infection.
[0012] (2) 5928-0054 can neutralize the main virulence factor of Staphylococcus aureus - hemolysin, and reduce the damage of inflammation to the body.
[0013] (3) 5928-0054 works by neutralizing bacterial virulence factors, reducing the bacterial mutation rate. Attached Figure Description
[0014] Figure 1 is a statistical chart of the hemolytic toxicity results of 5928-0054 on erythrocytes.
[0015] Figure 2 is a statistical graph showing the lysis results of red blood cells in the culture supernatant of Staphylococcus aureus USA300.
[0016] Figure 3 is a statistical chart showing the results of the inhibition of hemolytic activity of 5928-0054 on the culture supernatant of Staphylococcus aureus USA300.
[0017] Figure 4 is a statistical chart showing the results of the inhibition of hemolytic activity of Staphylococcus aureus USA300 hemolysin protein by 5928-0054.
[0018] Figure 5 is a statistical chart showing the results of the inhibition of hemolytic activity of Streptococcus suis SC19 hemolysin by 5928-0054.
[0019] Figure 6 is a schematic diagram of the molecular docking of Staphylococcus aureus α-hemolysin with 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 conformation of highest affinity between 6U49 and 5928-0054.
[0020] Figure 7 is a graph showing the caloric molar ratio of 5928-0054 to Staphylococcus aureus hemolysin.
[0021] Figure 8 is a kinetic reaction curve of 5928-0054 with Staphylococcus aureus hemolysin.
[0022] Figure 9 shows the survival curves of mice 5928-0054 infected with Staphylococcus aureus USA300 after treatment and without treatment.
[0023] Figure 10 shows the detection results of IL-6 and TNF-α in mouse serum.
[0024] Figure 11 shows the results of the biochemical level test in mouse blood.
[0025] Figure 12 shows the results of bacterial load detection in the lungs, spleen, kidneys, and liver of mice. Detailed Implementation
[0026] The following embodiments 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 embodiments are conventional means well known to those skilled in the art.
[0027] 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.
[0028] Example 1
[0029] (1) In a 96-well plate, 50 μL of 2% sheep red blood cells suspended in PBS were added to 50 μL of serially diluted 5928-0054 in PBS and incubated at 37°C for 1 hour to achieve final concentrations of 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. Wells with an equal volume of PBS added to the red blood cells served as negative controls, and 2.5% Triton X-100 served as positive controls. 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.
[0030] The results are shown in Figure 1. 5928-0054 itself does not cause hemolysis of red blood cells.
[0031] (2) Staphylococcus aureus USA300 was cultured at 37℃ for 12 h, centrifuged at 5000 r / min for 10 min at 4℃, and the supernatant was collected. Different volumes of culture supernatant were added to 2% sheep red blood cell phosphate buffer solution (PBS, pH=7.4) (total volume 1 mL) and incubated at 37℃ for 30 min. Finally, it was centrifuged at 500 r / min for 5 min at 4℃. Subsequently, 200 μL of supernatant was collected, and its OD value was measured at 543 nm. At the same time, the PBS solution containing 2% sheep red blood cells was treated with 2.5% Triton X-100 as a 100% positive control. The hemolytic activity of Staphylococcus aureus USA300 culture supernatant was evaluated by the ratio of the OD543 value of each sample to the positive control. The results are shown in Figure 2. When the culture supernatant of Staphylococcus aureus USA300 was 125 μL, about 90% of the red blood cells could be lysed.
[0032] (3) Staphylococcus aureus USA300 was cultured at 37℃ for 12 h, centrifuged at 5000 r / min for 10 min at 4℃, and the supernatant was collected. 125 μL of supernatant containing different concentrations of 5928-0054 was incubated at 37℃ for 30 min. Then, 875 μL of PBS buffer solution containing 2% sheep red blood cells was added, and the mixture was incubated at 37℃ for 30 min. Finally, the mixture was centrifuged at 1000 r / min for 5 min at 4℃, and 200 μL of the supernatant sample was collected. The OD543 value was measured. At the same time, the solution containing 2% sheep red blood cells was treated with 2.5% Triton X-100 as a positive control. The effect of 5928-0054 on the hemolytic activity of Staphylococcus aureus USA300 culture supernatant was evaluated by the ratio of the OD543 value of each sample to that of the positive control.
[0033] The results are shown in Figure 3. 5928-0054 significantly inhibited the hemolytic activity of Staphylococcus aureus USA300 supernatant in a concentration-dependent manner.
[0034] (4) The purified Staphylococcus aureus USA300 hemolysin (100 ng / mL) was co-incubated with different concentrations of 5928-0054, and the effect of 5928-0054 on the anti-Staphylococcus aureus hemolysin was evaluated according to the method in (3).
[0035] The results are shown in Figure 4, which are consistent with the results in (3). 5928-0054 inhibited the hemolytic activity of Staphylococcus aureus USA300 hemolysin.
[0036] (5) The purified Streptococcus suis SC19 hemolysin SLY (100 ng / mL) was co-incubated with different concentrations of 5928-0054, and the effect of 5928-0054 on the anti-Streptococcus suis SC19 hemolysin SLY was evaluated according to the method in (3).
[0037] The results are shown in Figure 5, indicating that 5928-0054 can also inhibit the hemolytic activity of Streptococcus suis hemolysin SLY.
[0038] Example 2
[0039] (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 structure of Staphylococcus aureus α-lysinin protein was obtained from the RCSB Database (https: / / www.rcsb.org / ). The lysinin protein (PDB ID: 6U49) is the 3D structure of the 2.35 Å lysin heptamer obtained by X-ray diffraction. Molecular docking was performed using MOE 2019 software to predict the interaction conformation between compound 5928-0054 and lysinin protein. The lysinin protein was completed and optimized using the QuickPrep module. Since the 6U49 structure contains specific ligands, all ligands, water molecules, and ADP were removed. Ligand preparation for compound 5928-0054 was performed by charge assignment, generating conformational isomers, and conformational optimization. The binding pocket of the lysinin protein was located using the Site finder module, and virtual particles were generated in the binding pocket.
[0040] The docking procedure used the Amber14:EHT force field to calculate the charges of all protein and ligand atoms. All ligand conformations were docked into docking pockets occupied by virtual particles. The triangle matcher method was used to position the ligands in the binding pockets, and 15 docking simulations were performed. The complex conformations were scored using the London dG function. The highest-scoring conformation between compounds 5928-0054 and 6U49 is shown in Figure 6A. Three sets of interactions were formed between 5928-0054 and 6U49, all of which were hydrogen bonds. In the hemolysin heptamer, hydrogen bonds were formed between 5928-0054 and the A chain A (ASN 172, LYS 154) and the B chain B (LYS 215) of 6U49 (Figure 6B). The highest energy was found between the O atom of 5928-0054 and the A chain LYS 154, at -7.8 kcal / mol, as illustrated in Figure 6C.
[0041] (2) The interaction between Staphylococcus aureus hemolysin and 5928-0054 was determined in vitro using isothermal calorimetric titration (ITC). Purified hemolysin and 5928-0054 were dissolved in PBS at pH 7.4 to final concentrations of 0.02 mmol / L and 0.2 mmol / L, respectively. The instrument was equilibrated to 25°C, and 50 μL of 5928-0054 was injected into a 300 μL sample cell containing purified hemolysin, 2.5 μL each time, with a reaction interval of 200 s. The molar ratio of heat between 5928-0054 and hemolysin during this reaction is shown in Figure 7. The kinetic reaction curve of 5928-0054 and hemolysin was fitted using NanoAnalyzer software, and the equilibrium dissociation constant (Kd) was calculated. The results are shown in Figure 8, and Kd was calculated to be 2.81 × 10⁻⁶. -7 The concentration of mol / L indicates that 5928-0054 has a good binding affinity with Staphylococcus aureus hemolysin.
[0042] This example demonstrates that 5928-0054 can also bind to hemolysins from other bacteria with structures similar to Staphylococcus aureus hemolysin, inhibiting their hemolytic activity.
[0043] Example 3
[0044] The mice used in the following experiments were seven-week-old female BALB / c mice.
[0045] (1) Survival rate experiment
[0046] Mice were randomly divided into two groups: a treatment group (5928-0054) and an untreated group, with 10 mice in each group. Overnight cultured Staphylococcus aureus USA300 was transferred to LB medium at a volume ratio of 1:100 and incubated at 37°C until OD600 = 0.5. The bacterial cells were collected after centrifugation at 4000 rpm for 5 min at 4°C, and resuspended in PBS for later use. In the survival rate experiment, each mouse was injected with 2.5 × 10⁻⁶ bacteria. 8 CFU bacterial count. Two hours after infection, mice were intraperitoneally injected with 5928-0054. The dosage was 5 mg / kg, administered every 12 hours for three consecutive days. The untreated group was injected with the same volume of PBS. The mouse survival curves constructed based on the experimental results are shown in Figure 9. Compared with the untreated group, the survival rate of mice in the 5928-0054 treatment group reached approximately 70%.
[0047] (2) Detection of inflammatory factors, blood biochemistry and tissue bacterial load
[0048] Mice were randomly divided into an untreated group, a 5928-0054 treatment group, and a blank control group, with 5 mice in each group. The untreated group and the 5928-0054 treatment group received intraperitoneal injections of a concentration of 5 × 10⁻⁶. 7CFU-infected mice (USA300) and mice in the blank control group were injected with the same volume of PBS. Two hours after infection, 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 injection, mice were sacrificed and blood and tissues were collected. The effects of 5928-0054 on blood biochemical (ALT, AST, CK) levels in USA300-infected mice were analyzed using a fully automated blood biochemistry analyzer. Furthermore, lungs, spleens, kidneys, and livers were ground, diluted, and inoculated onto LA plates, incubated overnight at 37°C, and bacteria were counted. The results were analyzed using a sensitive electrochemiluminescence platform (Quickplex, Meso-Scale Discovery). ® MSD was used to detect inflammatory cytokines. The results are as follows:
[0049] As shown in Figure 10, 5928-0054 can significantly inhibit the production of IL-6 and TNF-α in infected mice.
[0050] As shown in Figure 11, the 5928-0054 treatment significantly reduced blood biochemical levels in mice.
[0051] As shown in Figure 12, 5928-0054 can significantly reduce the bacterial load in the lungs, spleen, kidneys and liver of infected mice.
[0052] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. The application of compound 5928-0054 in the preparation of a drug for treating Staphylococcus aureus hemolysin, characterized in that: The structure of compound 5928-0054 is shown below:
2. The use of a pharmaceutically acceptable salt of compound 5928-0054 in the preparation of a drug for the treatment of Staphylococcus aureus hemolysin, characterized in that: The structure of compound 5928-0054 is shown below:
3. The use of compound 5928-0054 in the preparation of drugs for the prevention or treatment of Staphylococcus aureus infections, characterized in that: The structure of compound 5928-0054 is shown below:
4. The use of pharmaceutically acceptable salts of compound 5928-0054 in the preparation of medicaments for the prevention or treatment of Staphylococcus aureus infections, characterized in that: The structure of compound 5928-0054 is shown below:
5. The application of compound 5928-0054 in the preparation of drugs for antibacterial hemolysin, characterized in that: The structure of the compound 5928-0054 is shown below:
6. The use of a pharmaceutically acceptable salt of compound 5928-0054 in the preparation of a medicament for the production of an antibacterial hemolysin, characterized in that: The structure of compound 5928-0054 is shown below:
7. The use of compound 5928-0054 in the preparation of medicaments for the prevention or treatment of hemolysin-producing bacterial infections, characterized in that: The structure of the compound 5928-0054 is shown below:
8. The use of a pharmaceutically acceptable salt of compound 5928-0054 in the preparation of medicaments for the prevention or treatment of hemolysin-producing bacterial infections, characterized in that: The structure of compound 5928-0054 is shown below: