An α-hemolysin inhibitor and its application in the preparation of anti-infective drugs for Staphylococcus aureus.
By forming a stable complex between mangiferin and Hla protein, the problems of unstable inhibitor binding and insufficient affinity in existing technologies are solved, thus achieving effective inhibition of MRSA and reducing the bacterial load of Staphylococcus aureus infection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGXI UNIV
- Filing Date
- 2026-05-15
- Publication Date
- 2026-06-30
Smart Images
Figure CN122297458A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to a small molecule compound that can efficiently inhibit Staphylococcus aureus α-hemolysin (Hla), its pharmaceutical composition, and its pharmaceutical uses. Background Technology
[0002] Staphylococcus aureus is an important pathogenic bacterium, and its secreted α-hemolysin (Hla) is a key pore-forming toxin that causes cell lysis and leads to serious infections such as pneumonia, mastitis, and bacteremia. Currently, antibiotic treatment faces an increasingly serious challenge from drug resistance, especially the widespread prevalence of methicillin-resistant Staphylococcus aureus (MRSA) strains. Therefore, developing inhibitors that directly neutralize Hla virulence has become a new strategy for anti-infective therapy.
[0003] Existing technologies have reported antibody inhibitors and some small molecule inhibitors targeting Hla. However, antibody drugs suffer from drawbacks such as high production costs, the need for injection administration, and poor stability; while most known small molecule inhibitors bind reversibly to the Hla protein with low affinity, resulting in short-lasting inhibitory effects, and lack clear binding sites and mechanisms of action. Furthermore, existing technologies generally lack in vivo efficacy evidence in animal infection models (especially against MRSA-resistant strains) for directly reducing bacterial load in infected tissues. Therefore, there remains an urgent need to develop a small molecule inhibitor that can bind efficiently and stably to Hla, has a clearly defined site of action, provides sustained inhibitory effects, and demonstrates reliable in vivo efficacy. Summary of the Invention
[0004] To address the problems existing in the background art, the present invention provides an α-hemolysin inhibitor and its application in the preparation of Staphylococcus aureus anti-infective drugs.
[0005] To achieve the objectives of this invention, the following technical solution is adopted:
[0006] An α-hemolysin inhibitor, wherein the inhibitor is mangiferin.
[0007] Furthermore, the mangiferin forms a stable complex with Hla protein through various interactions such as hydrogen bonds and carbon-hydrogen bonds.
[0008] Furthermore, the interaction sites of the mangiferin with Hla protein include at least one of ARGA:200, THRA:199, LYSA:198, META:197, GLNA:194, TRPA:187, and TYRA:182.
[0009] Furthermore, the mangiferin blocks the pore-forming activity of Hla protein through irreversible or high-affinity binding, and the inhibitory effect is long-lasting.
[0010] The present invention also provides the use of an α-hemolysin inhibitor in the preparation of a drug that inhibits the activity of Staphylococcus aureus α-hemolysin.
[0011] The present invention also provides the use of an α-hemolysin inhibitor in the preparation of a drug for treating Staphylococcus aureus infection.
[0012] Furthermore, the Staphylococcus aureus includes the drug-resistant strain MRSA USA300.
[0013] Furthermore, mangiferin is the core active ingredient in the drug, and the hemolytic inhibitory activity of mangiferin against Hla protein is dose-dependent; the mangiferin can exert hemolytic inhibitory effects under both direct incubation and pre-incubation treatments.
[0014] The present invention also provides a pharmaceutical composition for treating Staphylococcus aureus infection, comprising the above-mentioned α-hemolysin inhibitor and pharmaceutically acceptable excipients.
[0015] Furthermore, the pharmaceutical composition is used to inhibit Staphylococcus aureus α-hemolysin-mediated cell lysis and is suitable for the prevention or treatment of infectious diseases caused by Staphylococcus aureus, including pneumonia, mastitis, and bacteremia.
[0016] Compared with the prior art, the present invention has the following technical advantages:
[0017] (1) This invention reveals for the first time through in vitro hemolysis test and molecular docking study that mangiferin can inhibit the hemolytic activity of Staphylococcus aureus supernatant and purified Hla protein in a dose-dependent manner, and interacts with multiple amino acid residues such as ARGA:200, THRA:199 and LYSA:198 of Hla protein by forming hydrogen bonds, thus clarifying its direct target and binding mechanism.
[0018] (2) The present invention has confirmed through red blood cell membrane washing experiment that the complex formed by mangiferin and Hla protein after pre-incubation can still effectively block hemolytic activity after repeated washing, indicating that mangiferin binds to Hla in a high affinity manner and has a long-lasting inhibitory effect.
[0019] (3) The present invention has demonstrated through a mouse mastitis model experiment that the mangiferin administration group (50 mg / kg) can significantly reduce the bacterial load in the mammary tissue of mice infected with the drug-resistant strain MRSA USA300 (P<0.01), providing the first direct pharmacodynamic evidence of mangiferin in vivo against drug-resistant Staphylococcus aureus infection. Attached Figure Description
[0020] Figure 1 This is a diagram of an in vitro hemolysis assay - direct incubation.
[0021] Figure 2 This is a diagram illustrating the specific implementation method of the in vitro hemolysis test - pre-incubation.
[0022] Figure 3 Hemolysis rate of Hla protein pre-incubated with different concentrations of mangiferin;
[0023] Figure 4 Figure showing the effect of mangiferin-Hla complex on the hemolytic activity of fresh erythrocytes after washing.
[0024] Figure 5 The diagram shows the molecular docking results of mangiferin and Hla.
[0025] Figure 6 This is a graph showing the bacterial load in mouse mastitis. Detailed Implementation
[0026] The present invention illustrates the compound mangiferin through the following examples, but the scope of the present invention is not limited thereto.
[0027] The mangiferin (MF) used was from the Aladdin brand, with a MW of 422.34 and a purity of 95%.
[0028] Example 1: Screening and Discovery of Hla Inhibitors
[0029] 1. Experimental materials and strains:
[0030] (1) The autoclaved TSB broth is cooled and set aside for later use;
[0031] (2) Mangiferin (MF) is from the Aladdin brand, with a MW of 422.34 and a purity of 95%;
[0032] (3) Weigh 0.500g of mangiferin powder into a centrifuge tube using a precision balance, add 10mL of DMSO to dissolve it, the concentration of mangiferin is 50mg / mL, vortex to mix and filter, pass the filtrate through a 0.22μm diameter filter membrane for physical sterilization, dispense into sterilized centrifuge tubes, and store in a -20℃ freezer in the dark.
[0033] (4) Test strain: Staphylococcus aureus strain MRSA USA300 was passaged on agar medium and cultured to a suitable size.
[0034] 2. Experimental methods and results:
[0035] (1) Inoculate Staphylococcus aureus in a centrifuge tube containing 3.5 mL TSB broth, place it in a shaker at 37°C and shake at 220 rpm until the late logarithmic growth phase, then centrifuge and remove the bacterial supernatant.
[0036] (2) Mangiferin and bacterial supernatant were incubated together in a 1.5 mL centrifuge tube to prepare a 500 μL system. The insufficient part was made up with PBS. The system was divided into two groups. One group was mixed with rabbit red blood cells and incubated directly at 37°C for 35 min. The other group was prepared by incubating the drug and bacterial supernatant at 37°C for 35 min before adding rabbit red blood cells and mixing well. The mixture was then incubated at 37°C for another 35 min.
[0037] (3) The group without bacterial supernatant and drug was used as a negative control, and the group without drug was used as a positive control;
[0038] (4) After the incubation is over, centrifuge at 3500 rpm for 3 min, take 200 μL of supernatant and add it to a 96-well plate, measure its absorbance at 543 nm and calculate the hemolysis rate.
[0039] Hemolysis rate calculation formula: Hemolysis rate (%) = (Experimental group absorbance - Negative control group absorbance) / (Positive control group absorbance - Negative control group absorbance) × 100%
[0040] The results are as follows Figure 1 , Figure 2 As shown, mangiferin exhibited certain inhibitory activity against hemolysis under both treatment methods, with its stability and potential efficacy being more prominent. Therefore, it was identified as a target drug for further in-depth research.
[0041] Example 2: Expression and purification of Staphylococcus aureus α-hemolysin (Hla) protein
[0042] 1. Experimental materials and strains:
[0043] (1) Host bacteria: BL21(DE3) Escherichia coli;
[0044] (2) Recombinant plasmid: Pczn1-hla; (Core gene source: hla protein gene of Staphylococcus aureus strain USA300, vector contains 6×His tag, resistance is Amp)
[0045] (3) The TB broth, which has been autoclaved, is cooled and set aside for later use;
[0046] (4) Isopropyl-β-D-thiogalactopyranoside (IPTG) is a Solarbio brand product;
[0047] (5) The His-tagged protein purification kit is from Beyotime.
[0048] (6) The PBS buffer (pH 7.4) was from the Sewell brand;
[0049] (7) The 10kDa molecular weight cutoff ultrafiltration tube is from the Beyotime brand;
[0050] (8) Weigh 0.2383g of IPTG using a precision balance and dissolve it in 8mL of sterile deionized water. Make up to 10mL and then filter it through a 0.22μm filter membrane for sterilization. After dispensing, store it at -20℃ in the dark to obtain a 100mM IPTG solution.
[0051] 2. Induction and purification of Hla protein:
[0052] (1) BL21(DE3)-hla was passaged on agar medium containing ampicillin resistance and cultured to the appropriate size;
[0053] (2) Inoculate BL21(DE3)-hla into a centrifuge tube containing 3.5 mL of ampicillin-resistant TB broth and place it in a shaker at 37°C and shake at 220 rpm overnight;
[0054] (3) BL21(DE3)-hla was transferred at a 1:100 ratio and cultured in 200 mL of TB medium containing ampicillin-resistant culture medium until od. 600 When the concentration is 0.6~0.8, add 0.2 mM IPTG and incubate at 16℃ and 180 rpm for 16 h.
[0055] (4) Collect the bacterial cells by centrifugation at 4℃ and 8000rpm for 10min, and resuspend them three times with PBS;
[0056] (5) Ultrasonic disruption, centrifugation at 4℃ and 12000rpm for 20min, and collection of supernatant;
[0057] (6) Equilibration: Add 1 mL of equilibration solution to His tag affinity resin, centrifuge at 10000 rpm for 1 min, and discard the effluent;
[0058] (7) Mix the balanced affinity resin with the crushed supernatant and shake at 4°C for 1 hour to allow it to fully combine;
[0059] (8) Gravity column preparation: Place the gravity column from the kit onto the support;
[0060] (9) Sample loading: Slowly add the supernatant of the above-mentioned bacterial cell lysis to the gravity column, allowing the supernatant to flow down the column wall and drip naturally (control the flow rate to 1-2 drops / second to ensure that the target protein is fully bound to the resin), and collect the effluent (for use in detecting residual impurities).
[0061] (10) Washing: Add 5 column volumes of the washing buffer provided with the kit, let it drip naturally to wash away unbound proteins until the effluent is clear (samples can be taken for testing to confirm that the proteins have been basically removed), then discard the effluent;
[0062] (11) Target protein elution: Add 5 column volumes of the elution buffer provided with the kit, let it drip naturally, and collect all the elution solution.
[0063] (12) Ultrafiltration tube equilibration: Add 1 mL of PBS buffer (pH 7.4) to a 10 kDa ultrafiltration tube, centrifuge at 4000 g for 5 min at 4 °C, discard the liquid in the lower chamber, and retain the liquid in the upper chamber;
[0064] (13) Concentration and replacement: Add all the eluent collected by the gravity column to the upper chamber of the ultrafiltration tube, centrifuge at 4°C and 4000g for 15 min until about 100 μL of liquid remains in the tube (target protein concentration); then add 1 mL of PBS buffer to the upper chamber of the ultrafiltration tube, repeat centrifugation twice to completely replace the imidazole in the eluent, and finally collect the concentrated liquid in the upper chamber.
[0065] Example 3: Verification of inhibitory activity against purified Hla protein
[0066] 1. Experimental methods and results:
[0067] (1) The purified Hla protein in Example 2 was pre-incubated with different concentrations of mangiferin and then neutralized.
[0068] (2) For example Figure 3 The results showed that mangiferin can directly inhibit the hemolytic activity of Hla protein in a dose-dependent manner.
[0069] Example 4: Combining stability verification (membrane washing experiment)
[0070] 1. Experimental methods and results:
[0071] (1) Incubate Hla with the drug (0.2 mg / mL) or an equal volume of DMSO at 37°C for 35 min;
[0072] (2) Add 2% red blood cells and mix, then incubate at 37°C for 30 min;
[0073] (3) Centrifuge the above mixture at 4°C and 3500 rpm for 3 min, and carefully discard the supernatant. Resuspend the red blood cell pellet in pre-cooled PBS and wash 3 times to completely remove unbound components;
[0074] (4) Resuspend the washed red blood cells in 500 μL PBS and add 2% fresh red blood cells. Incubate at 37°C for 35 min.
[0075] (5) After the incubation is over, centrifuge at 3500 rpm for 3 min, take 200 μL of supernatant and add it to a 96-well plate, measure its absorbance at 543 nm and calculate the hemolysis rate.
[0076] Hemolysis rate calculation formula: Hemolysis rate (%) = (Experimental group absorbance - Negative control group absorbance) / (Positive control group absorbance - Negative control group absorbance) × 100%
[0077] Depend on Figure 4 It was found that in the erythrocyte membrane washing experiment, after the drug mangiferin was pre-incubated to form a complex with Hla protein, it was incubated with the first batch of erythrocytes and washed to remove unbound components. Then, a second batch of fresh erythrocytes was added for hemolysis assay. The results showed that compared with the positive control group (Hla + fresh erythrocytes), the hemolysis rate of the sample group (drug + Hla pre-incubation followed by washing) was significantly reduced. This indicates that the complex formed by the drug and Hla after pre-incubation could not cause significant hemolysis after washing. This suggests that the drug may inhibit the pore-breaking activity of Hla through irreversible or high-affinity binding, thus preventing it from lysing erythrocytes. The DMSO solvent control group did not show significant interference.
[0078] Example 5: Molecular docking research
[0079] 1. Experimental materials and software:
[0080] (1) Protein structure: Hla protein (PDB number: 6U49), three-dimensional structure data were obtained from the Protein Database (PDB). This protein is the core object for studying drug targets.
[0081] (2) Target compound: mangiferin: PubChem CID is 5281647. Three-dimensional structure data were obtained from the PubChem database as a potential active compound to be studied.
[0082] (3) Molecular docking software: Discovery Studio software was selected, which has functions such as molecular docking and interaction analysis, and can realize the docking simulation of compounds and proteins and the analysis of binding sites and interaction types;
[0083] 2. Experimental methods and results:
[0084] (1) Using Discovery Studio software, molecular docking simulations were performed between mangiferin and Hla protein (PDB:6U49) to analyze their interaction with Hla protein. The specific results are as follows:
[0085] (2) For example Figure 5 It can be seen that the docking results of mangiferin and Hla protein show that mangiferin and Hla protein have multiple interactions:
[0086] Hydrogen bonding: Mangiferin molecules form conventional hydrogen bonds with amino acid residues such as ARGA:200 and GLNA:194 of Hla protein, as well as carbon-hydrogen bonds with residues such as TYRA:182. These hydrogen bonding interactions help stabilize the binding conformation of mangiferin and protein.
[0087] Distribution of key sites: The binding region involves amino acid sites such as ARGA:200, THRA:199, LYSA:198, META:197, GLNA:194, TRPA:187, and TYRA:182, suggesting that these sites may be key regions for mangiferin to inhibit the hemolytic activity of Hla protein.
[0088] Example 6: Effect of mangiferin on bacterial load of Staphylococcus aureus-infected mice with mastitis
[0089] 1. Experimental materials and instruments:
[0090] (1) Experimental animals: SPF-grade female KM mice, 6-8 weeks old, weighing 20-22g, were housed in a constant temperature (22±2℃) and constant humidity (50±5%) environment with free access to food and water. After 3 days of acclimatization, they were used for the experiment.
[0091] (2) Test strain: Staphylococcus aureus MRSA USA300 (same as in Example 1), inoculated into TSB broth, cultured at 37°C with shaking at 220 rpm until the logarithmic growth phase, and the bacterial concentration was adjusted to 1×10⁻⁶ with sterile PBS. 7 CFU / mL, for later use;
[0092] (3) Test drug: mangiferin, dissolved in DMSO and diluted with sterile physiological saline to prepare a drug solution with a concentration of 100 mg / mL (final concentration of DMSO ≤ 5%, no obvious toxicity), and used immediately after preparation.
[0093] 2. Test methods:
[0094] (1) After anesthetizing mice with 1.25% tribromoethanol (15mg / kg) via intraperitoneal injection, they were fixed on the operating table and the skin of the mammary region was disinfected.
[0095] (2) Draw 50 μL of LMRSA USA300 bacterial solution (1×10⁻⁶) into a sterile microsyringe. 7 CFU / mouse was slowly injected into the fourth pair of mammary fat pads of mice to establish an acute mastitis model.
[0096] (3) The negative control was injected with an equal volume of sterile PBS;
[0097] (4) Grouping and administration: After successful modeling, the mice were randomly divided into 4 groups of 4 mice each;
[0098] (5) Model control group: After modeling, an equal volume of sterile saline was injected into the peritoneum;
[0099] (6) Mangiferin administration group: Mangiferin administration solution (50 mg / kg) was injected intraperitoneally after modeling.
[0100] (7) Negative control group: Intraperitoneal injection of an equal volume of sterile saline;
[0101] (8) Solvent control group: intraperitoneal injection of an equal volume of DMSO;
[0102] (9) After 24 hours, the mice were euthanized by cervical dislocation, and the fourth pair of infected mammary gland tissues were dissected under sterile conditions and the surface bloodstains were washed off with sterile PBS.
[0103] (10) Bacterial count determination: The breast tissue homogenate was serially diluted 10-fold, and 100 μL of homogenate from each dilution was evenly spread on mannitol high-salt agar plates. The plates were placed in a constant temperature incubator at 37℃ and incubated upside down for 16 h. The number of colonies (CFU) on the plates was counted, and plates with a colony count between 30 and 300 were selected for calculation.
[0104] Formula for calculating bacterial count: Bacterial count (CFU / g) = (number of colonies on a plate × dilution factor) / weight of breast tissue (g).
[0105] 3. Experimental Results:
[0106] like Figure 6 As shown, the bacterial count in the mammary gland tissue of the mangiferin-treated group was significantly reduced to (0.7±0.15)×10⁻⁶. 5 The CFU / g level was significantly different from that of the model control group (P<0.01), indicating that mangiferin is effective in reducing the number of bacteria in the mammary tissue of mice infected with Staphylococcus aureus.
Claims
1. An alpha-hemolysin inhibitor, characterized in that, The inhibitor is mangiferin.
2. The α-hemolysin inhibitor according to claim 1, characterized in that, The mangiferin forms a stable complex with Hla protein through various interactions such as hydrogen bonds and carbon-hydrogen bonds.
3. The α-hemolysin inhibitor according to claim 2, characterized in that, The binding sites of mangiferin and Hla protein include at least one of ARGA:200, THRA:199, LYSA:198, META:197, GLNA:194, TRPA:187, and TYRA:
182.
4. The α-hemolysin inhibitor according to claim 1, characterized in that, The mangiferin blocks the pore-forming activity of Hla protein through irreversible or high-affinity binding, and the inhibitory effect is long-lasting.
5. The use of an α-hemolysin inhibitor according to any one of claims 1-4 in the preparation of a medicament for inhibiting the activity of Staphylococcus aureus α-hemolysin.
6. The use of an α-hemolysin inhibitor according to any one of claims 1-4 in the preparation of a medicament for treating Staphylococcus aureus infection.
7. The application according to claim 6, characterized in that, The Staphylococcus aureus includes the drug-resistant strain MRSAUSA300.
8. The application according to claim 5 or 6, characterized in that, The drug contains mangiferin as its core active ingredient, and the hemolytic inhibitory activity of mangiferin against Hla protein is dose-dependent; the mangiferin can exert hemolytic inhibitory effects under both direct incubation and pre-incubation treatments.
9. A pharmaceutical composition for treating Staphylococcus aureus infection, characterized in that, It comprises the α-hemolysin inhibitor according to any one of claims 1-4, and pharmaceutically acceptable excipients.
10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition is used to inhibit Staphylococcus aureus α-hemolysin-mediated cell lysis and is suitable for the prevention or treatment of infectious diseases caused by Staphylococcus aureus, including pneumonia, mastitis, and bacteremia.