Medical application of daphnetin in preparation of Acinetobacter baumannii polyphosphate kinase inhibitor

Abstract

The invention provides a medical application of daphnetin in preparation of a PPK1 inhibitor. A growth curve test, a PPK1 enzyme activity inhibition test, a bacterial polyphosphate content determination test, a bacterial biofilm inhibition test and a hydrogen peroxide stimulation and thermal stimulation test show that the daphnetin can reduce the resistance mechanism of the acinetobacter baumannii to external stress factors by inhibiting the PPK1 enzyme activity. The invention provides a new way and a potential lead compound for preventing and controlling acinetobacter baumannii infection.

Description

Technical Field

[0001] The invention discloses a new use of daphnetin, relates to the medical use of daphnetin in preparing an inhibitor of Acinetobacter baumannii polyphosphate kinase 1 (PoLyphosphate kinase 1, PPK1), and belongs to the technical field of medical pharmaceuticals. Background Art

[0002] Acinetobacter baumannii is a non-fermenting Gram-negative bacillus with strong environmental adaptability and clonal propagation capabilities, capable of surviving for months in dry environments. It is a major pathogen of hospital-acquired infections, capable of long-term survival in hospital environments and possessing strong adhesion, causing a variety of illnesses, including hospital-acquired pneumonia, bacteremia, wound site infections, and urinary tract infections. It has been designated by the World Health Organization as a "priority pathogen" for which new treatments are urgently needed. The multidrug- and extensively drug-resistant nature of Acinetobacter baumannii poses a global public health threat. Its complex resistance mechanisms complicate clinical drug selection, posing a major challenge in clinical treatment.

[0003] PPK is a key enzyme regulating the synthesis of bacterial polyphosphates (poLyphosphates, poLyP). Widely conserved in bacteria, it is an ideal target for anti-infective therapy. Studies have shown that PPK catalyzes the conversion of ATP from bacterial ATP to poLyP. This polyphosphate is involved in bacterial biofilm formation, bacterial motility, bacterial persistence, antibiotic susceptibility, host cell invasion and adhesion, and can enhance bacterial adaptability to environmental stresses such as ultraviolet radiation, strong oxidation, and hypoxia. Therefore, inhibiting PPK activity in Acinetobacter baumannii is of great significance for the prevention and treatment of A. baumannii infections. Summary of the Invention

[0004] The present invention provides a medical use of daphnetin in the preparation of a PPK1 inhibitor, discloses that daphnetin can inhibit the activity of PPK1, and provides an effective target drug for preventing and treating Acinetobacter baumannii infection.

[0005] The daphnetin of the present invention has the molecular formula: C

[0006] The present invention shows that daphnetin can reduce the tolerance of Acinetobacter baumannii by inhibiting PPK1 enzyme activity through growth curve test, PPK1 enzyme activity inhibition test, bacterial polyphosphate content determination test, bacterial biofilm inhibition test, hydrogen peroxide stimulation and heat stimulation test. The Acinetobacter baumannii used in the test is The positive effects of the present invention are: A new medical use of daphnetin in the preparation of PPK1 inhibitors is provided, and it is disclosed that daphnetin can inhibit the activity of PPK1, providing a new approach and potential lead compound for the prevention and treatment of Acinetobacter baumannii infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 These are the growth curves of the wild strain (WT) and the PPK1-deficient strain (Δppk1::Apr) before and after treatment with different concentrations of daphnetin in the present invention.

[0008] Figure 2 This is a graph showing the inhibition of PPK1 enzyme activity by daphnetin of the present invention.

[0009] Figure 3 This is the determination of poLyP content in WT and Δppk1::Apr before and after treatment with daphnetin of the present invention.

[0010] Figure 4 Quantitative images of biofilm staining produced by WT and Δppk1::Apr before and after treatment with daphnetin of the present invention.

[0011] Figure 5 This is a quantitative graph showing the number of surviving bacteria stimulated by hydrogen peroxide in WT and Δppk1::Apr before and after treatment with daphnetin of the present invention.

[0012] Figure 6 This is a quantitative graph showing the number of WT and Δppk1::Apr surviving heat stimulation before and after treatment with daphnetin of the present invention. DETAILED DESCRIPTION

[0013] The present invention is further described by way of examples below, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or alteration of the present invention that can be easily implemented by a person skilled in the art will fall within the scope of the claims of the present invention.

[0014] Example 1 Daphnetin can be used as a PPK1 inhibitor in any pharmaceutically acceptable carrier.

[0015] Example 2 Daphnetin can be used as a PPK1 inhibitor for preparing drugs for treating infectious diseases.

[0016] Example 3 Daphnetin is used as a PPK1 inhibitor to treat infectious diseases caused by bacteria.

[0017] Test Example 1 Growth curve determination WT and Δppk1::Apr glycerol strains were inoculated into LB medium overnight (37°C, 220 rpm). The next day, the overnight culture suspension was spread into 20 mL of LB medium at a 1:100 ratio and cultured until the OD600 nm reached 0.3. Groups were divided into WT, Δppk1::Apr, and WT treated with different concentrations of daphnetin. The optical density absorbance at 600 nm (OD600 nm) of the bacterial samples was measured every 1 hour.

[0018] Conclusion: The loss of PPK1 does not affect the growth of Acinetobacter baumannii. Daphnetin does not inhibit the growth of Acinetobacter baumannii when the concentration is ≤64 μg / mL. Figure 1 .

[0019] Test Example 2 PPK1 enzyme activity inhibition assay PPK1 can catalyze the formation of polyphosphates from ATP in vitro, and DAPI can be used to detect polyphosphate formation. Based on this property, 1 μg of PPK1 protein and different concentrations of daphnetin (0 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, and 64 μg / mL) were added to an enzyme activity inhibition system (50 mM Hepes, pH 7.5, 50 mM ammonium sulfate, and 5 mM magnesium chloride) and incubated at room temperature for 15 minutes. 1 mM ATP and 40 μM DAPI were added, and after vortexing and mixing, the cells were further incubated in the dark for 10 minutes. Polyphosphate content was measured at 430 / 550 (excitation / emission) wavelengths. The experimental group was treated with different concentrations of daphnetin, while the positive reaction group was treated with an equal volume of PBS. The drug inhibition rate was calculated according to the following formula: Inhibition rate (%) = (1 - fluorescence value of the experimental group / fluorescence value of the positive reaction group) × 100% Conclusion: With the increase of daphnetin concentration, the activity of PPK1 enzyme was significantly inhibited. Figure 2 .

[0020] Test Example 3 Determination of polyphosphate content in bacteria Overnight cultures of WT and Δppk1::Apr bacteria were expanded in 20 mL of LB medium at a 1:100 ratio. Experimental groups included WT, Δppk1::Apr, and WT treated with different concentrations of daphnetin (4 μg / mL, 8 μg / mL, 16 μg / mL, and 32 μg / mL). These cultures were cultured to an OD600 nm of 0.6. 2 mL of each aliquot was collected by centrifugation. The cells were resuspended in an equal volume of low-phosphate MOPS medium and incubated at 37°C for 2 h to stimulate poLyP synthesis. The cells were then collected by centrifugation, washed twice with 50 mM Hepes buffer, and resuspended in 2 mL of 50 mM Hepes buffer. Heat the bacterial suspension in a 60°C water bath for 10 minutes, collect the bacteria by centrifugation, and resuspend them in 2 mL of DAPI assay buffer. After vortexing, add 10 μM DAPI, vortex and mix thoroughly, and let stand at room temperature for 10 minutes in the dark. Pipette 200 μL of the sample into a 96-well plate and measure the polyphosphate content of the bacteria at 420 / 550 nm (excitation / emission wavelengths).

[0021] Conclusion: Daphnetin can effectively reduce the polyphosphate content in bacteria. The polyphosphate content in WT treated with 32 μg / mL daphnetin was significantly reduced. Figure 3 .

[0022] Test Example 4 Bacterial biofilm test Acinetobacter baumannii can develop adaptive protective mechanisms by synthesizing biofilms. The inhibitory effect of daphnetin on biofilm formation was systematically evaluated using in vitro quantitative analysis. The experiments involved untreated bacteria (WT, Δppk1::Apr) and WT treated with 8, 16, or 32 μg / mL daphnetin. Overnight cultures of WT and Δppk1::Apr were adjusted to an OD600 nm of 0.1 and added to LB medium at a ratio of 1:500. The treated cells were then treated with the corresponding concentrations of daphnetin, shaken to mix, plated into 24-well plates, and incubated in a 30°C incubator for 2 days. The plates were then removed, rinsed twice with PBS, oven-dried, and stained with 0.1% crystal violet solution for 1 hour. The plates were then decolorized with glacial acetic acid, and the absorbance at 570 nm was measured using a spectrophotometer.

[0023] Conclusion: The biofilm formed by the PPK1-deficient strain was significantly reduced compared with the WT strain. In addition, daphnetin (16 μg / mL, 32 μg / mL) could significantly inhibit the biofilm formation of WT. Figure 4 .

[0024] Test Example 5 Hydrogen peroxide stimulation test Overnight cultures of WT and Δppk1::Apr were inoculated into LB medium at a ratio of 1:100. Experimental groups included untreated bacteria (WT, Δppk1::Apr) and WT treated with 16 μg / mL and 32 μg / mL daphnetin. The cultures were then incubated (37°C, 200 rpm) until an OD600 nm of 0.6. One mL of the bacterial culture was aspirated and centrifuged in a 1.5 mL centrifuge tube. The bacteria were suspended in an equal volume of PBS containing 0.05 mM H2O2 and incubated for 30 minutes. The cells were centrifuged again, the supernatant aspirated, and the bacteria were suspended in an equal volume of PBS and vortexed to mix. Serial dilutions were then plated onto solid LB medium to determine the number of viable bacteria.

[0025] Conclusion: The bacterial survival rate of the PPK1-deficient strain was significantly lower than that of the WT strain. Daphnetin can effectively reduce the bacterial tolerance to hydrogen peroxide stimulation. Figure 5 .

[0026] Test Example 6 Thermal stimulation test Overnight cultures of WT and Δppk1::Apr were inoculated into LB medium at a ratio of 1:100. Experimental groups included untreated bacteria (WT, Δppk1::Apr) and WT treated with 16 μg / mL and 32 μg / mL daphnetin. These samples were cultured to an OD600 nm of 0.6, then centrifuged and the supernatant discarded. The bacteria were resuspended in an equal volume of PBS and heat-stimulated at 60°C for 20 minutes. The cells were then diluted and plated onto LB solid medium to determine the number of surviving bacteria.

[0027] Conclusion: The bacterial survival rate of the PPK1-deficient strain was significantly lower than that of the WT strain. Daphnetin can effectively reduce the bacterial tolerance to heat stimulation. Figure 6 .

Claims

1. The medical use of daphnetin in the preparation of Acinetobacter baumannii polyphosphate kinase inhibitors.

2. The use according to claim 1, characterized in that The medical use refers to that daphnetin inhibits the biological function of Acinetobacter baumannii polyphosphate kinase.

3. The use according to claim 1, characterized in that The Acinetobacter baumannii inhibitor can effectively inhibit the formation of Acinetobacter baumannii biofilm and reduce its resistance to hydrogen peroxide and heat stimulation.

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