Application of emodin in carbapenem-resistant Klebsiella pneumoniae

Abstract

This invention relates to the application of emodin in the treatment of carbapenem-resistant Klebsiella pneumoniae, belonging to the field of pharmaceutical technology. This invention provides the use of emodin in the preparation of products resistant to carbapenem-resistant Klebsiella pneumoniae. Emodin binds to KPC-2 type carbapenemase produced by carbapenem-resistant Klebsiella pneumoniae, inhibiting the activity of KPC-2 in hydrolyzing carbapenems, thereby enhancing the bactericidal effect of the antibiotics. Studies have shown that emodin can bind to the TRP-105 and THR-237 sites of the KPC-2 protein, altering the functional group composition and secondary structure of the KPC-2 protein, thereby inhibiting KPC-2 hydrolytic activity and enhancing the bactericidal effect of the antibiotics. This invention expands the medicinal value of emodin and provides a novel combination therapy regimen for the treatment of carbapenem-resistant Klebsiella pneumoniae infections.

Description

Technical Field

[0001] This invention relates to the application of emodin in carbapenem-resistant Klebsiella pneumoniae, and belongs to the field of pharmaceutical technology. Background Technology

[0002] For a long time, carbapenem antibiotics have been considered the last line of defense against Gram-negative bacteria. However, with the increase in the overuse and abuse of antimicrobial drugs in recent years, bacteria have gradually evolved resistance to antibiotics. Multidrug-resistant bacteria have emerged frequently in recent years and have become an important global public health problem. Among them, carbapenem-resistant Klebsiella pneumoniae (CRKP) is a key bacterium to be controlled in hospital infections and poses a huge threat to global public health.

[0003] KPC (Klebsiella pneumoniae carbapenemases) is a carbapenemase, belonging to class A enzymes (serine carbapenemases). It is the most prevalent carbapenemase in Enterobacteriaceae worldwide, especially Klebsiella pneumoniae. KPC-2 and KPC-3 are the most common. In my country, more than 70% of carbapenem-resistant Klebsiella pneumoniae produce KPC-2. The activities of KPC-2 and KPC-3 can be inactivated or inhibited by new β-lactamase inhibitors such as avibactam, faborbactam, and lelibactam.

[0004] my country boasts abundant plant-derived natural product resources, which are increasingly favored by researchers due to their wide availability, high safety, and broad antibacterial spectrum. Numerous studies have demonstrated that plant-derived natural products, used alone or in combination with antibiotics, have a significant effect on inhibiting bacterial growth and reproduction. Furthermore, plant-derived natural products can effectively reduce antibiotic usage and side effects, delay the development of drug resistance, and are of great significance to human health and medical advancement. Emodin is an anthraquinone derivative extracted from rhubarb. It possesses antibacterial, antitumor, anti-inflammatory, and anti-angiogenic effects, showing broad application potential in the pharmaceutical field. Currently, research on the effects of emodin against carbapenem-resistant Klebsiella pneumoniae has not yet emerged. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to alleviate the antibiotic resistance problem caused by KPC-2 positive drug-resistant Klebsiella pneumoniae, thereby providing an application of emodin in carbapenem-resistant Klebsiella pneumoniae.

[0006] Therefore, the present invention provides the following technical solution: This invention provides the use of emodin in the preparation of products resistant to carbapenem antibiotics against Klebsiella pneumoniae.

[0007] In one embodiment of the present invention, the product includes an inhibitor, a reagent, or a drug.

[0008] In one embodiment of the present invention, the carbapenem-resistant Klebsiella pneumoniae includes at least one of ATCC® BAA1705™ and clinically isolated Klebsiella pneumoniae.

[0009] In one embodiment of the present invention, the clinically isolated Klebsiella pneumoniae includes at least one of 2024CCZX-055 and 2024CCZX-060.

[0010] In one embodiment of the present invention, the carbapenem antibiotics include meropenem, imipenem, or biapenem.

[0011] In one embodiment of the present invention, the emodin enhances the bactericidal effect of antibiotics by disrupting the formation of biofilms in carbapenem-resistant Klebsiella pneumoniae.

[0012] In one embodiment of the present invention, when disrupting the formation of carbapenem-resistant Klebsiella pneumoniae biofilm, the mass ratio of emodin to carbapenem antibiotics is 2:1.

[0013] In one embodiment of the present invention, the emodin enhances the bactericidal effect of the antibiotic by binding to KPC-2 carbapenemase produced by carbapenem-resistant Klebsiella pneumoniae and inhibiting the activity of KPC-2 in hydrolyzing carbapenem antibiotics.

[0014] In one embodiment of the present invention, the emodin inhibits the activity of KPC-2 in hydrolyzing carbapenem antibiotics by altering the functional group composition and secondary structure of the KPC-2 protein, thereby enhancing the bactericidal effect of the antibiotics.

[0015] In one embodiment of the present invention, molecular docking, molecular dynamics simulation, and surface plasmon resonance (SPR) analysis showed that the emodin can effectively bind to the KPC-2 protein.

[0016] In one embodiment of the present invention, Fourier transform infrared spectroscopy and circular dichroism analysis showed that the emodin inhibited the activity of KPC-2 in hydrolyzing carbapenem antibiotics by changing the functional group composition and secondary structure of KPC-2 protein, thereby enhancing the bactericidal effect of antibiotics, but the expression level of KPC-2 protein was not affected.

[0017] In one embodiment of the present invention, the key binding sites of the emodin to the KPC-2 protein are TRP-105 and THR-237.

[0018] In one embodiment of the present invention, emodin can enhance the in vitro antibacterial effect of meropenem against carbapenem-resistant Klebsiella pneumoniae and its in vivo therapeutic effect in mice.

[0019] The present invention also provides an antibacterial agent for inhibiting carbapenem-resistant Klebsiella pneumoniae, wherein the active ingredients of the antibacterial agent include rhein and carbapenem antibiotics.

[0020] In one embodiment of the present invention, the emodin has a synergistic effect with carbapenem antibiotics, and the emodin can enhance the bactericidal effect of the antibiotics.

[0021] In one embodiment of the present invention, the carbapenem antibiotics include meropenem, imipenem, or biapenem.

[0022] In one embodiment of the present invention, the dosages of emodin and meropenem in the antibacterial agent are 12.5-25 mg / kg and 5 mg / kg, respectively.

[0023] In one embodiment of the present invention, emodin and meropenem have a synergistic antibacterial effect against Klebsiella pneumoniae.

[0024] In one embodiment of the present invention, emodin and meropenem have a synergistic antibacterial effect against Klebsiella pneumoniae, with a synergistic index (FIC) of 0.3 to 0.7.

[0025] In one embodiment of the present invention, emodin and meropenem have a synergistic antibacterial effect against Klebsiella pneumoniae, with a synergistic index (FIC) of 0.3 to 0.5.

[0026] In one embodiment of the present invention, the MIC value of emodin against Klebsiella pneumoniae ATCC® BAA1705™ is 64 μg / mL, the MIC value of meropenem against Klebsiella pneumoniae ATCC® BAA1705™ is 32 μg / mL, and the fractional inhibitory concentration index (FIC) of the two used in combination is 0.5.

[0027] In one embodiment of the present invention, emodin and meropenem have a synergistic antibacterial effect against clinically isolated Klebsiella pneumoniae 2024CCZX-055, with a synergistic index FIC of 0.375.

[0028] In one embodiment of the present invention, emodin and meropenem have a synergistic antibacterial effect against clinically isolated Klebsiella pneumoniae 2024CCZX-060, with a synergistic index FIC of 0.3125.

[0029] The present invention also provides the use of the above-mentioned antibacterial agent in the preparation of a medicament for treating pneumonia caused by KPC-2 positive bacteria.

[0030] In one embodiment of the present invention, the bacteria include Klebsiella pneumoniae and Escherichia coli.

[0031] The technical solution of this invention has the following advantages: 1. This invention provides the use of emodin in the preparation of products against carbapenem-resistant Klebsiella pneumoniae, wherein the carbapenem-resistant Klebsiella pneumoniae includes at least one of ATCC® BAA1705™ and clinically isolated Klebsiella pneumoniae. This study shows that emodin inhibits the activity of KPC-2 in hydrolyzing carbapenems by binding to KPC-2 type carbapenemase, thereby restoring the bactericidal effect of the antibiotics. Emodin can also inhibit the activity of KPC-2 in hydrolyzing carbapenems and enhance the bactericidal effect by binding to the TRP-105 and THR-237 sites of the KPC-2 protein, thereby altering the functional group composition and secondary structure of the KPC-2 protein. This invention expands the medicinal value of emodin and provides a novel combination therapy regimen for treating carbapenem-resistant Klebsiella pneumoniae infections.

[0032] Furthermore, emodin enhances the bactericidal effect of antibiotics by disrupting the formation of biofilms in carbapenem-resistant Klebsiella pneumoniae. Bacterial biofilms, also known as bacterial biofilms, are large aggregates of bacteria that adhere to a contact surface and secrete polysaccharide matrices, fibrin, lipid proteins, etc., enveloping themselves within them. Once formed, biofilms possess natural resistance to antibiotics and the body's immune system. Even with high doses of antibiotics, it is difficult for them to reach the biofilm, and they can only act on the surface of the biofilm or on free-floating bacteria in the blood that cause infection. Emodin enhances the bactericidal effect of antibiotics by directly killing bacteria, disrupting the integrity of bacterial cells, and destroying the biofilm formed by carbapenem-resistant Klebsiella pneumoniae.

[0033] 2. This invention also provides an antibacterial agent for inhibiting carbapenem-resistant Klebsiella pneumoniae, wherein the active ingredients of the antibacterial agent include emodin and carbapenem antibiotics. Emodin in the antibacterial agent has a synergistic effect with carbapenem antibiotics, and emodin can enhance the bactericidal effect of the antibiotics. The dosages of emodin and meropenem in the antibacterial agent are 12.5-25 mg / kg and 5 mg / kg, respectively. Combined treatment with emodin and meropenem can alleviate pulmonary edema in mice, and the bacterial load in the lungs of mice after combined treatment is significantly lower than in other treatment groups, indicating that the antibacterial agent provided in this application shows good therapeutic effects on mice infected with Klebsiella pneumoniae. Attached Figure Description

[0034] Figure 1 3D schematic diagram of the docking of emodin with KPC-2; Figure 2 3D schematic diagram of meropenem docking with KPC-2; Figure 3 2D schematic diagram of the docking of emodin with KPC-2; Figure 4 2D schematic diagram of meropenem docking with KPC-2; Figure 5 Figures showing the molecular dynamics simulation results of emodin, meropenem and KPC-2; (A)-(C) Root mean square deviation (RMSD) of protein, ligand and complex system; (D) Root mean square fluctuation (RMSF); Figure 6 Figures showing the molecular dynamics simulation results of emodin, meropenem, and KPC-2; (A) radius of gyration (Rg); (B) solvent accessible surface area (SASA); Figure 7 Figures showing the molecular dynamics simulation results of emodin, meropenem, and KPC-2; (A) Decomposition diagram of the binding energy of emodin (MMGBSA) in molecular dynamics simulation; (B) Decomposition diagram of the binding energy of meropenem (MMGBSA) in molecular dynamics simulation; Figure 8 The image shows the pET28a-KPC-2 WT plasmid from Example 3. Figure 9 The image shows the pET28a-KPC-2 MT plasmid from Example 3. Figure 10 The results show the protein purification of the pET28a-KPC-2 WT plasmid in Example 3; Figure 11 The results show the protein purification of the pET28a-KPC-2 MT plasmid in Example 3; Figure 12SPR kinetics analysis of the binding of different concentrations of emodin to KPC-2 protein (WT type and MT type); (A) KPC-2 (WT); (B) KPC-2 (MT-TRP-105); (C) KPC-2 (MT-THR-237); Figure 13 Fourier transform infrared spectra of KPC-2 protein before and after emodin treatment; Figure 14 Circular dichroisms of KPC-2 protein before and after emodin treatment; Figure 15 Immunoblot and grayscale analysis of KPC-2 protein after treatment with different concentrations of emodin; (A) Treatment for 4 h; (B) Treatment for 8 h; Figure 16 The effect of emodin on the relative activity of KPC-2 protein; Figure 17 Lineweaver-Burk curves showing the inhibition of KPC-2 protein activity by emodin; Figure 18 Antibacterial chessboard diagram of emodin and meropenem combined; Figure 19 bactericidal curve of emodin combined with meropenem; Figure 20 Staining results of live and dead bacteria under the combined action of emodin and meropenem; (A) Control; (B) Meropenem; (C) Emodin; (D) Emodin combined with meropenem; Scale bar, 10 μm; Figure 21 Figure 1. Semi-quantitative adhesion experiment results of crystal violet on biological membranes; Figure 22 Three-dimensional images of biofilms obtained by confocal microscopy in live-dead staining experiments; (A) Control; (B) Meropenem; Scale bar, 200 μm; Figure 23 Three-dimensional images of biofilms obtained by confocal microscopy in live-dead staining experiments; (A) emodin; (B) emodin combined with meropenem; scale bar, 200 μm; Figure 24 Results of rhein cytotoxicity assay; Figure 25 Figures showing the results of weight monitoring, wet / dry weight ratio of lung tissue, and lung colony count in pneumonia model mice; (A) Weight monitoring of pneumonia model mice; (B) Wet / dry weight ratio of lung tissue; (C) Lung colony count. Detailed Implementation

[0035] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0036] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0037] The materials and instruments used in the following embodiments are shown in the table below: Table 1. Main Experimental Materials and Reagents

[0038] Table 2. Main Instruments and Equipment

[0039] Klebsiella pneumoniae ATCC® BAA1705™: purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). Clinical isolations of Klebsiella pneumoniae 2024CCZX-055 and 2024CCZX-060: provided by Jilin Provincial Center for Disease Control and Prevention.

[0040] Example 1: Molecular docking analysis of the interaction between emodin and KPC-2 protein 1. Test Methods Simulations were performed using the protein-ligand docking software Autodock Vina 1.2.2. The molecular structures of emodin and meropenem were obtained from the PubChem compound database; the KPC-2 protein structure was downloaded from the PDB database. Molecular docking simulations were performed using Autodock Vina 1.2.2, and visualization was performed using Pymol 3.0.3 software. Specifically, all protein and molecular files were converted to PDBQT format, all water molecules were removed, and polar hydrogen atoms were added. The grid was centered to cover the domains of each protein and accommodate free molecular motion.

[0041] 2. Test Results It is generally believed that molecular docking energies below -7.0 kcal / mol exhibit strong binding activity. A schematic diagram of the 3D results of molecular docking between emodin, meropenem, and KPC-2 protein is shown (e.g., Figure 1 and Figure 2As shown in the figure, the binding energy of emodin, meropenem and KPC-2 protein is -8.6 kcal / mol, indicating that there is a strong interaction between emodin, meropenem and KPC-2 protein.

[0042] A schematic diagram of the 2D results of molecular docking of emodin, meropenem, and KPC-2 protein is shown (e.g.) Figure 3 and Figure 4 As shown, emodin can form hydrogen bonds and hydrophobic interactions with key residues of the KPC-2 protein, such as SER-70, TRP-105, and THR-237. These interactions stabilize emodin within the active pocket of KPC-2, effectively preventing meropenem from binding to KPC-2.

[0043] Example 2: Molecular dynamics simulation analysis of the interaction between emodin and KPC-2 protein 1. Test Methods Small molecule pretreatment was performed using AmberTooLs22 to add a GAFF force field, while Gaussian 16W was used for hydrogenation and RESP potential calculations. The potential data were added to the molecular dynamics system topology file. Simulations were conducted at a static temperature of 300 K and atmospheric pressure (1 Bar), using Amber99sb-iLdn as the force field and water molecules as the solvent (Tip3p water model). An appropriate amount of Na was added to further refine the process. + Ions were used to neutralize the total charge of the simulated system. The molecular dynamics simulation system first employed the steepest descent method for energy minimization, followed by 100,000 steps of isothermal-isochoric (NVT) and isothermal-isobaric (NPT) ensemble equilibrium, with a coupling constant of 0.1 ps and a duration of 100 ps. Finally, a free molecular dynamics simulation was run, consisting of 5,000,000 steps at a step size of 2 fs, lasting 100 ns. After the simulation, the software's built-in tools were used to analyze the trajectories, calculating the root mean square variance (RMSD), root mean square fluctuation (RMSF), and protein radius of gyration (Rg) for each amino acid trajectory, combined with data such as free energy (MMGBSA).

[0044] 2. Test Results Root mean square deviation (RMSD) is a good indicator of conformational stability of proteins and ligands, and also measures the degree of deviation of atomic positions from their initial positions. The smaller the deviation, the better the conformational stability. Figure 5As shown in (A), RMSD analysis of the ligands themselves reveals that the average RMSD of emodin-KPC-2 is only 0.03 ± 0.01 nm, while that of meropenem-KPC-2 is 0.09 ± 0.01 nm, indicating that emodin is more stable within the binding site. RMSD fluctuations in proteins can be used to compare changes in protein conformation after ligand binding. Figure 5 (B) As shown, after binding to small molecules, the RMSD curve of the protein tends to stabilize rapidly and remain at a low level, indicating that the complex structure is stable. Figure 5 As shown in (C), the protein-small molecule complex system tends to reach equilibrium at around 20 ns with minimal fluctuations, indicating that the system is relatively stable overall. The fluctuation curves of the emodin-KPC-2 protein complex and the meropenem-KPC-2 protein complex show some differences, but the fluctuation amplitude still exhibits high stability.

[0045] Root mean square fluctuations (RMSF) can represent the flexibility of amino acid residues in a protein. For example... Figure 5 As shown in (D), in both the emodin-KPC-2 protein complex and the meropenem-KPC-2 protein complex, the amino acid residues at positions 100-110, 150-170, and 230-240 exhibit greater flexibility compared to amino acid residues at other positions. This is consistent with the results of the binding sites in the molecular docking results, which further indicates that the amino acid sites for the binding of emodin, meropenem, and KPC-2 protein exist in these more flexible amino acid residues.

[0046] The radius of gyration (Rg) can be used to describe changes in the overall structure and characterize the compactness of protein structures; a larger change in Rg indicates a more expanded system. Figure 6 (A) shows that the Rg values ​​of the complex system first increase and then decrease, and then stabilize, with the two values ​​being close. This indicates that the target protein-small molecule complex first expands and then contracts during the simulation, and then reaches a stable state.

[0047] Solvent-accessible surface area (SASA) is an indicator for assessing protein surface area. For example... Figure 6 As shown in (B), the small molecule-KPC-2 system exhibits slight fluctuations in the first 80 ns and gradually stabilizes. This indicates that the binding of emodin and meropenem to KPC-2 affects the binding microenvironment and leads to some changes in SASA.

[0048] The amino acid residue binding free energy decomposition (MMGBSA) can represent the contribution of protein amino acids to the bonding of small molecules, such as... Figure 7As shown in (A), (B) and Table 3, the total binding free energy of emodin to KPC-2 protein and the amino acid residues that contribute the most to the energy are TRP-105 and THR-237. Site-directed mutagenesis will be performed on these two amino acid sites to further verify the key amino acid sites of the interaction between emodin and KPC-2 protein.

[0049] In summary, the binding of emodin to KPC-2 protein affects the protein structure, making it more compact, thus demonstrating a good binding effect between emodin and KPC-2 protein. Furthermore, located in the active pocket where meropenem binds to KPC-2, emodin effectively prevents the binding of meropenem to KPC-2.

[0050] Table 3. Molecular dynamics simulation of MMGBSA binding energy decomposition of emodin, meropenem and KPC-2

[0051] Example 3: Site-directed mutagenesis of amino acid sites and protein isolation and purification 1. Test Methods Site-directed mutagenesis at amino acid sites: Using pET28a-KPC-2 (WT) plasmid (a gift from Professor Deng Xuming of the College of Veterinary Medicine, Jilin University; see reference "Characterization of Corosolic Acid as a KPC-2 Inhibitor That Increases the Susceptibility of KPC-2-Positive Bacteria to Carbapenems") as the DNA template, the plasmid map is shown below. Figure 8 As shown in the figure, the insert sequence in the spectrum is as shown in SEQ ID NO.1; 50 ng of DNA template, 2 μL each of forward and reverse primers (as shown in Table 4 below) (10 μmol / L), and 25 μL of high-fidelity enzyme 2×PrimeSTAR MaxPremix (Takara) were added to a 50 μL PCR system, and the remaining volume in the system was made up with ultrapure water. DNA amplification was performed using a PCR instrument, and the program was as follows: ① Pre-denaturation: 95 ℃, 1 min → ② Denaturation: 98 ℃, 10 s → ③ Annealing: 55 ℃, 5 s → ④ Extension: 72 ℃, 1 min (30 cycles of ②-④) → ⑤ Second extension: 72 ℃, 5 min → Hold at 4 ℃.

[0052] Table 4. Upstream and downstream primer sequences

[0053] The amplification products were digested with DpnI enzyme to reduce false positives caused by template DNA. The specific procedure was as follows: 1 μL of DpnI enzyme was added per 50 μL of water, and the mixture was incubated at 37 °C for 1–2 h, followed by inactivation at 70 °C for 15 min. The PCR product was then purified using a PCR product purification kit and transformed into competent E. coli cells. Positive colonies obtained were sequenced to identify the constructed mutant pET28a-KPC-2 (MT).

[0054] Protein isolation and purification: Activated *E. coli* BL21(DE3)-pET28a-KPC-2 (WT) or *E. coli* BL21(DE3)-pET28a-KPC-2 (MT) were inoculated into LB medium with a final kanamycin sulfate concentration of 50 μg / mL at a ratio of 1:100, and the OD of the culture medium was measured. 600nm The absorbance when OD 600nm When the concentration reached 0.6, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.7 mM to induce protein expression in *E. coli*. The cells were cultured overnight at 16 °C, collected by centrifugation, resuspended in PBS, and sonicated. The supernatant from the lysed cells was purified by affinity chromatography using a nickel column to remove protein impurities and unbound proteins, yielding pure KPC-2 protein. After dialysis, the protein was stored at -80 °C. The purified protein was analyzed by SDS-PAGE electrophoresis and Coomassie brilliant blue staining.

[0055] 2. Test Results Figures 8-9 The diagram shows the pET28a-KPC-2 WT and MT expression vectors. The pET-28a-KPC-2 (WT) recombinant plasmid was sequenced for verification. Sequencing results were analyzed using BLAST data from the NCBI database. The results showed that the target fragment had 99% similarity to the KPC-2 gene sequence reported in the NCBI database, indicating almost complete identity. Using the plasmid as a template, site-directed mutagenesis was performed. Positive clones were selected and sequenced to identify the constructed mutant pET28a-KPC-2 (MT).

[0056] The purified proteins were analyzed using SDS-PAGE electrophoresis and Coomassie brilliant blue staining, such as... Figure 10 , Figure 11 As shown, a distinct protein expression band appeared at a molecular weight of around 30 kDa, which is similar in size to the reported KPC-2 protein (31.1 kDa), indicating that the target protein was successfully expressed in the pET28a-KPC-2 (WT) recombinant plasmid and the pET28a-KPC-2 (MT) mutant plasmid.

[0057] Example 4: Surface plasmon resonance (SPR) analysis of the interaction between emodin and KPC-2 protein 1. Test Methods Proteins were immobilized on the surface of a CM5 chip. DMSO solutions with final concentrations of 4.5% and 6% were prepared using PBS and 100% DMSO. Calibration curves were then constructed using DMSO concentrations of 4.5%, 5.0%, 5.5%, and 6.0%. The DMSO solution containing emodin was diluted 20-fold with PBS buffer to reduce the DMSO content from 100% to 5%. Different concentrations of emodin were sequentially injected into the experimental and reference channels at a flow rate of 30 μL / min, with binding and dissociation times of 60 s. Both binding and dissociation steps were performed using the running reagents.

[0058] Affinity fitting was performed using Biacore 8K analysis software (Biacore Insight Evaluation Software), and the KD value of the samples was calculated.

[0059] 2. Test Results The kinetics of the interaction between emodin and KPC-2 were determined using SPR, such as... Figure 12 As shown in (A), after passing different concentrations of emodin through a chip immobilized with KPC-2 protein, the SPR refractive index of the chip surface changed in a dose-dependent manner. A smaller equilibrium dissociation constant (KD) indicates better binding between the compound and the target protein and stronger affinity. The results show that emodin can bind to KPC-2 protein, with an equilibrium dissociation constant (KD) of 2.44 × 10⁻⁶. -5 M exhibits strong affinity.

[0060] Site-directed mutagenesis was performed on the amino acid sites of the KPC-2 protein. The isolated and purified KPC-2 (MT type) protein was then immobilized on the surface of a CM5 chip, and SPR experiments were performed according to the steps described above. Figure 12 As shown in (B) and (C), after mutations at the TRP105 and THR237 sites, the equilibrium dissociation constant (KD) increased significantly, reaching 6.94 × 10⁻⁶. -4 M, 1.73×10 -4 M reflects a decrease in substrate affinity. This further confirms that TRP-105 and THR-237 are key amino acid residues for emodin's competitive binding with MEM and KPC-2.

[0061] Example 5: Fourier transform infrared spectroscopy analysis of the effect of emodin treatment on KPC-2 protein 1. Test Methods KPC-2 protein levels before and after treatment with 32 μg / mL emodin were recorded using Fourier transform infrared (FTIR) spectroscopy at 4000–500 cm⁻¹. -1 FTIR spectra within the range, with a resolution of 2 cm⁻¹ -1 The obtained data were analyzed and plotted using Origin 2023 to reveal the effects of emodin treatment on the functional groups of the KPC-2 protein.

[0062] 2. Test Results The results are as follows Figure 13 As shown, it is located at 3357 cm. -1 The broad peak at 3073 cm⁻¹ is mainly due to the OH stretching vibration in the amide A region, primarily originating from OH groups in the KPC-2 protein's side chain / main chain and water molecules. -1 The peak is mainly due to the stretching vibration of NH in the amide A region, at 2949 cm⁻¹. -1 and 2881 cm -1 The values ​​represent the antisymmetric and symmetric stretching vibrations of CH in the methyl (-CH3) and methylene (-CH2-) groups, respectively. The value is located at 1629 cm⁻¹. -1 This is the stretching vibration peak of C=O in the amide I region, which mainly includes α-helices (1650~1660 cm⁻¹). -1 ), β-fold (1620~1640 cm) -1 ), β-turn (1660~1680 cm) -1 ) and random curls (1640~1650 cm) -1 ), located in the amide II zone (1480~1570 cm). -1 ) 1533 cm -1 The peak at 1464 cm⁻¹ is mainly a superposition of CN stretching and NH bending vibration peaks. -1 The peak is mainly composed of the bending vibration of CH, located at 1297 cm⁻¹. -1 The peak is mainly composed of CN stretching vibrations, located at 1036 cm⁻¹. -1 The peak is mainly a stretching vibration peak of CO, located at 1152 cm⁻¹ in the spectrum after treatment with emodin. - A distinct new peak for CO appears at ¹, primarily because the vibrational signal of the phenolic hydroxyl functional group of emodin is superimposed on the KPC-2 protein spectrum, and is located at 862 cm⁻¹. - The appearance of a stretching vibration peak of CH on the benzene ring at position ¹ indicates the addition of emodin to the modified protein, while a peak is also observed at 3357 cm⁻¹. - ¹ and 3073 cm -The stretching vibration peaks of the hydroxyl and amino groups (OH / NH) at position ¹ broaden and even shift, mainly because hydrogen bonds form between the phenolic hydroxyl groups of emodin and the amino groups of the KPC-2 protein, leading to peak broadening and a red shift. Meanwhile, the peak at 1629 cm⁻¹... -1 The intensity of the C=O stretching vibration peak at 1036 cm⁻¹ is enhanced, mainly because C=O is also present in emodin, leading to an increase in the relative content of C=O in the modified substance. Furthermore, emodin binds to the carbonyl group of the KPC-2 protein through hydrogen bonds or hydrophobic interactions, altering the vibrational frequency of the C=O bond and thus increasing its peak intensity. -1 The CO peak intensity at that location was significantly enhanced, and it blue-shifted to 1076 cm⁻¹. -1 This is mainly because the phenolic hydroxyl groups (-OH) in the anthraquinone structure of emodin form a hydrogen bond network with the hydroxyl groups of the KPC-2 protein glycan chain, increasing the electron cloud density of the CO bonds and the vibrational bond strength, thus causing the peak position to shift to a higher wavenumber. Furthermore, the CO vibration of the free glycan chain (1036 cm⁻¹) -1 They mostly exhibit a random coil conformation, and after binding with emodin, they form a rigid β-glycosidic bond structure (1070~1080 cm⁻¹). -1 ).

[0063] Example 6: Circular dichroism analysis of the effect of emodin treatment on KPC-2 protein 1. Test Methods The circular dichroism spectra of KPC-2 protein in the wavelength range of 190 to 250 nm were recorded using a CD spectrophotometer before and after treatment with 32 μg / mL emodin. The recorded spectra were analyzed using CDNN software to compare the changes in the secondary structure of KPC-2 protein before and after emodin treatment.

[0064] 2. Test Results The results are as follows Figure 14 As shown, emodin-induced KPC-2 underwent significant conformational changes, with an increased proportion of β-sheets and β-turns, and a decreased proportion of α-helices and irregular coils.

[0065] Example 7: Western blotting analysis of the effect of emodin treatment on KPC-2 protein 1. Test Methods Western blotting was used to detect the potential impact of drugs on target protein expression. Single colonies of Klebsiella pneumoniae ATCC® BAA1705™ were picked from culture dishes and inoculated into 5 mL of sterile LB medium. The cultures were then placed in a temperature-controlled shaking incubator at 37 °C and a shaking speed of 180 r / min for overnight incubation. The following day, the bacterial culture was adjusted to OD200. 600nm0.1 was reserved. Subsequently, different concentrations of emodin (0, 2, 8, and 32 μg / mL) were added to the bacterial suspension, and the mixture was co-cultured at 37 ℃ for 4 h and 8 h. 500 μL of the bacterial suspension was centrifuged, resuspended in 1× Loading buffer, and boiled in a metal bath at 100 ℃ for 5 min. The sample was then loaded for SDS-PAGE electrophoresis with the following parameters: 80 V, 30 min for stacking gel, 120 V, 60 min for separating gel. The sample was then transferred to a polyvinylidene fluoride (PVDF) membrane, blocked with skim milk powder at room temperature for 1 h, washed 2-3 times with TBST, incubated overnight at 4 ℃ with primary antibody, washed 2-3 times with TBST, added HRP-labeled goat anti-mouse secondary antibody, incubated at room temperature for 1 h, washed 2-3 times with TBST, and then ECL developing solution was added. The membrane was then exposed and observed in a chemiluminescence imaging system, and the grayscale value of each band was analyzed using ImageJ software.

[0066] Data processing methods: Statistical analysis was performed using GraphPad Prism 8.0 software. One-way ANOVA and Tukey's multiple comparison test were used to analyze significant differences between groups. Data are expressed as mean ± standard deviation (SD), with the mean being the average of three repeated experiments. A p-value < 0.05 was considered statistically significant. * indicates p < 0.05, ** indicates p < 0.01.

[0067] 2. Test Results like Figure 15 As shown in (A) and (B), under different concentrations of emodin, there was no significant difference in the grayscale ratio between the target protein band and the reference band, and the ratio did not decrease with prolonged drug treatment time, indicating that the effect of emodin on KPC-2 expression is not concentration- or time-dependent. Emodin interacts with KPC-2, affecting its protein conformation and consequently its antibiotic hydrolytic activity, but does not affect KPC-2 protein expression.

[0068] Example 8: Analysis of the effect of cefotaxime hydrolysis experiment on the activity of emodin on KPC-2 protein 1. Test Methods The inhibitory activity of emodin on KPC-2 protein was investigated using a cefotaxime hydrolysis assay. Purified KPC-2 protein and different concentrations of emodin were sequentially added to 190 μL of PBS buffer in 96-well plates to achieve final emodin concentrations of 0, 2, 4, 8, 16, and 32 μg / mL, and a final KPC-2 protein concentration of 20 μg / mL. After thorough mixing and incubation at room temperature for 20 minutes, 10 μL of cefotaxime was added to achieve a final concentration of 10 μg / mL, and the plate was incubated at 37 °C for 10 minutes. The absorbance at 492 nm was then measured using a microplate reader and recorded as A1. A negative control group (containing only the drug and no protein) was established to eliminate interference from drug color; its absorbance was recorded as A2. A positive control group (containing no drug) was established, with a relative KPC-2 activity of 100% and its absorbance recorded as A3.

[0069] The formula for calculating KPC-2 protein activity is as follows: KPC-2 enzyme activity (%) = × 100%, KPC-2 enzyme inhibition rate (%) = 1 - KPC-2 enzyme activity (%).

[0070] 2. Test Results like Figure 16 As shown, emodin exhibited an inhibition rate greater than 50% at a concentration of 4 μg / mL, IC50 50 The value was 6.604 μg / mL.

[0071] Example 9: Inhibitory kinetics of emodin on KPC-2 protein 1. Test Methods At a protein concentration of 20 μg / mL, different concentrations of meropenem and emodin were added, resulting in final meropenem concentrations of 100, 80, 60, 40, and 20 μM, and final emodin concentrations of 32, 16, 8, 4, and 0 μg / mL. After incubation at 37 ℃ in the dark for 30 min, the protein was analyzed using OD0.05. 300nm The rate of change reflects the hydrolysis rate under different conditions. A Lineweaver-Burk plot is generated by plotting the substrate concentration (1 / S) versus the reciprocal of the reaction rate (1 / V) to determine the inhibition type and calculate K. m and V max .

[0072] 2. Test Results The slope of the inhibition kinetic curve represents K. m / V max Value, intercept represents 1 / V max Value. For example... Figure 17 As shown, V max The slope remains constant, but increases with increasing emodin concentration, i.e., K...m The increase in the Michaelis constant indicates that emodin inhibits KPC-2 protein activity by competitively binding to the active site with meropenem.

[0073] Example 10: Determination of MIC for single-drug susceptibility testing of emodin and meropenem 1. Test Methods According to the Clinical and Laboratory Standards Institute (CLSI) guidelines, the antibacterial activity of emodin was determined using the broth microdilution method, and the specific procedure is as follows: The overnight cultured Klebsiella pneumoniae ATCC® BAA1705™ was diluted to a concentration of 1×10⁻⁶ using MH broth medium. 6 CFU / mL, emodin was dissolved in cell-grade DMSO, filtered through a 0.22 μm filter, and then serially diluted in MH broth to 16, 32, 64, 128, 256, 512, 1024, and 2048 μg / mL. 100 μL of bacterial culture was inoculated into a 96-well plate, followed by 100 μL of emodin solution at a gradient concentration, resulting in final emodin concentrations of 8, 16, 32, 64, 128, 256, 512, and 1024 μg / mL. 100 μL of bacterial culture + 100 μL of MH broth was used as a positive control, and 200 μL of MH broth was used as a negative control. After incubating the 96-well plates at 37 ℃ in the dark for 24 hours, the lowest concentration of emodin in the group without visible turbidity was visually observed and considered the MIC value of emodin against Klebsiella pneumoniae ATCC® BAA1705™. The method for determining the MIC value of meropenem against Klebsiella pneumoniae ATCC® BAA1705™ is the same as above.

[0074] 2. Test Results The MIC value of emodin against Klebsiella pneumoniae ATCC® BAA1705™ was 64 μg / mL, and the MIC value of meropenem against Klebsiella pneumoniae ATCC® BAA1705™ was 32 μg / mL. Example 11: Combined drug susceptibility test of emodin and meropenem 1. Test Methods The checkerboard method involves serially diluting the medium in a horizontal row from left to right and vertical column from top to bottom, based on the minimum inhibitory concentration (MIC) test, to form a 96-well plate checkerboard. The synergistic effect of emodin and meropenem against Klebsiella pneumoniae ATCC® BAA1705™ is then determined by the turbidity of the culture medium in each treatment well.

[0075] Synergistic effects are determined based on the fractional inhibitory concentration (FIC) index, calculated using the formula: FIC index = (MIC) / (FIC * (MIC * (FIC ...))))))))))))) A药联用 / MIC A药单用 ) + (MIC B药联用 / MIC B药单用 ) A FIC index ≤ 0.5 indicates a synergistic effect; 0.5 < FIC index ≤ 1 indicates an additive effect; 1 < FIC index ≤ 2 indicates no relationship; and a FIC index greater than 2 indicates an antagonistic effect.

[0076] 2. Test Results like Figure 18 As shown, emodin and meropenem exhibit synergistic antibacterial activity against Klebsiella pneumoniae ATCC® BAA1705™, clinically isolated Klebsiella pneumoniae 2024CCZX-055, and 2024CCZX-060, with synergistic indices FIC of 0.5, 0.375, and 0.3125, respectively.

[0077] Example 12: Time-bacterial killing curves of emodin and meropenem 1. Test Methods Pick a single colony from a petri dish and inoculate it with *Klebsiella pneumoniae* ATCC® BAA1705™ in 5 mL of fresh LB broth. Fix the colony in a shaking incubator at 37 °C and 180 r / min, and incubate overnight. Adjust the bacterial concentration to 1 × 10⁻⁶. 5 CFU / mL of emodin, meropenem, emodin + meropenem, and MH medium without any drugs were added separately and co-cultured at 37 ℃. 100 μL samples were taken at each set time point (0 h, 2 h, 4 h, 8 h, 12 h, 16 h, 24 h), appropriately diluted with sterile PBS, and 10 μL was spread on NA plates and incubated overnight at 37 ℃. Colony counting was performed the next day, and a time-sterilization curve was plotted by plotting the logarithm of time versus colony count.

[0078] 2. Test Results like Figure 19 As shown, MEM represents the meropenem group. The change trend of colony count over time was quantified by the time-bactericidal curve. The combined treatment of emodin and meropenem showed a time-dependent bactericidal effect on bacteria, while the drug-only treatment groups did not show significant inhibitory or bactericidal effects on bacteria, and the colony count increased significantly over time.

[0079] Example 13: Staining of live and dead bacteria treated with emodin and meropenem 1. Test Methods The overnight cultured Klebsiella pneumoniae ATCC® BAA1705™ was diluted to a concentration of 5 × 10⁻⁶ using MH broth medium. 8 CFU / mL; emodin (16 μg / mL), meropenem (8 μg / mL), emodin (16 μg / mL) + meropenem (8 μg / mL) and MH medium without any drugs were added respectively. After incubation in a constant temperature shaker at 37 ℃ for 2 h, live / dead bacteria were stained in the dark using a live / dead bacteria double staining kit (SYTO 9 / PI Live / Dead Bacterial Double Stain Kit). The survival of bacteria after different treatments was observed using a laser confocal microscope.

[0080] 2. Test Results like Figure 20 As shown in (A) to (D), MEM represents the meropenem group ( Figure 20 (B) in the middle group and (B) in the control group. Figure 20 The control group (A) exhibited abundant green fluorescence with virtually no red fluorescence, and its activity was better than that of the drug-treated group. Figure 20 Compared to (B) and (C) groups, the group treated with emodin and meropenem ( Figure 20 The significant increase in red fluorescence in the middle (D) indicates that emodin can enhance the bactericidal effect of meropenem.

[0081] Example 14: Semi-quantitative adhesion experiment of biofilm with crystal violet and experiment on live and dead biofilm staining 1. Test Methods The overnight cultured Klebsiella pneumoniae ATCC® BAA1705™ was diluted to a concentration of 1×10⁻⁶ using MH broth medium. 6 CFU / mL, 2 mL of bacterial culture was inoculated into a 24-well plate and incubated at 37°C for 48 h. The supernatant was discarded, and MH medium containing emodin (16 μg / mL), meropenem (8 μg / mL), or emodin (16 μg / mL) + meropenem (8 μg / mL) and no other drugs was added. The plate was incubated at 37°C for 2 h. Afterward, the plate was gently washed three times with 0.9% sterile saline. Each well was stained with 0.1% crystal violet solution for 15 min, followed by three more washes with 0.9% sterile saline to remove excess stain. The plate was then air-dried and photographed. Finally, 1 mL of 33% glacial acetic acid was added to each well to dissolve the crystal violet, and the OD was measured. 595nm The absorbance was measured at the point.

[0082] The bacterial suspension at the above concentration was inoculated into confocal microplates and cultured for 48 h to form a biofilm. After the bacterial suspension was formed, the suspension was discarded, and MH medium containing emodin, meropenem, emodin + meropenem, and no drugs was added. The plates were then incubated at 37 ℃ for 12 h and the supernatant was discarded. The plates were washed three times with 0.9% sterile physiological saline. 500 μL of bacterial live / dead staining solution prepared with physiological saline was added to the center of the plates, and the three-dimensional biofilm structure was observed using a confocal microscope.

[0083] Data processing methods: Statistical analysis was performed using GraphPad Prism 8.0 software. One-way ANOVA and Tukey's multiple comparison test were used to analyze significant differences between groups. Data are expressed as mean ± standard deviation (SD), where the mean is the average of three repeated experiments. A p-value < 0.05 was considered statistically significant. * indicates p < 0.05, ** indicates p < 0.01.

[0084] 2. Test Results like Figure 21 As shown, MEM represents the meropenem group, and the OD of the co-treatment group is... 595nm The value decreased significantly, indicating that emodin can enhance the destruction of already formed biofilms by meropenem.

[0085] like Figures 22-23 The three-dimensional image of the biofilm is shown, with the control group ( Figure 22 Both (A) and (B) showed a distinct dark green color, indicating that the biofilm was relatively intact, the biological activity was good, and it was similar to emodin (A). Figure 23 (A) , Meropenem ( Figure 22 Compared with the single-use group (B), the combined-use group (B) Figure 23 The intensity of red fluorescence in (B) was significantly enhanced, while the intensity of green fluorescence was weakened, which further demonstrates that emodin can enhance the biofilm clearance ability of meropenem.

[0086] Example 15: Effect of emodin on cell viability as determined by MTT assay 1. Test Methods Adjust L02 cells to 1×10 4At a concentration of 100 μL / mL, cells were seeded into each well of a 96-well cell culture plate. After complete cell adhesion, the culture medium was removed, and 200 μL of DMEM containing different concentrations of emodin were added to achieve final emodin concentrations of 0, 1, 2, 4, 8, and 16 μg / mL. After 24 h of incubation, 20 μL of 5 mg / mL MTT reagent was added to each well, and the plate was incubated for another 4 hours. The culture medium was then discarded, and 150 μL of DMSO was added to each well. The absorbance at 570 nm was measured to calculate cell viability.

[0087] Data processing methods: Statistical analysis was performed using GraphPad Prism 8.0 software. One-way ANOVA and Tukey's multiple comparison test were used to analyze significant differences between groups. Data are expressed as mean ± standard deviation (SD), where the mean is the average of three repeated experiments. A p-value < 0.05 was considered statistically significant. * indicates p < 0.05, ** indicates p < 0.01.

[0088] 2. Test Results like Figure 24 As shown, when the concentration of emodin reached 16 μg / mL, it had no significant effect on the viability of L02 cells.

[0089] Example 16: The therapeutic effect of combined emodin and meropenem on KPC-2 positive Klebsiella pneumoniae pneumonia 1. Test Methods Six- to eight-week-old female BALB / c mice (purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were randomly divided into six groups. Except for the healthy group mice which had normal diet and water, the other groups of mice were used to establish a mouse Klebsiella pneumoniae ATCC® BAA1705™ pneumonia infection model.

[0090] Each mouse was anesthetized by inhalation of ether until it was semi-conscious. The mouse's head was held upright with one hand, exposing both nasal cavities. 50 μL of solution containing 1.0 × 10⁻⁶ ppm was evenly instilled. 8CFU (Chemical Fusion) of Klebsiella pneumoniae suspension. One hour after infection, mice in the meropenem treatment group were subcutaneously injected with meropenem (MEM, 10 mg / kg, twice daily); mice in the low-dose emodin treatment group were subcutaneously injected with emodin (Emodin-L, 25 mg / kg, twice daily); mice in the high-dose emodin treatment group were subcutaneously injected with emodin (Emodin-H, 50 mg / kg, twice daily); mice in the low-dose combination treatment group (Combination-L, meropenem 5 mg / kg + emodin 12.5 mg / kg); mice in the high-dose combination treatment group (Combination-H, meropenem 5 mg / kg + emodin 25 mg / kg); and mice in the control and model groups were subcutaneously injected with sterile water for injection. Body weight changes in each group were recorded daily. After 72 hours, mice were euthanized by cervical dislocation, and lung tissue was collected and divided into two portions. One portion was homogenized for colony counting. The other portion was weighed and stored at 60°C for several days until the lung tissue dried, and the wet-to-dry weight ratio of the lung was calculated.

[0091] Data processing methods: Statistical analysis was performed using GraphPad Prism 8.0 software. One-way ANOVA and Tukey's multiple comparison test were used to analyze significant differences between groups. Data are expressed as mean ± standard deviation (SD), where the mean is the average of three repeated experiments. A p-value < 0.05 was considered statistically significant. * indicates p < 0.05, ** indicates p < 0.01.

[0092] 2. Test Results Following infection with Klebsiella pneumoniae, mice exhibited rapid breathing, poor mental state, disheveled fur, significantly reduced activity, and often huddled in the corner of their cages. Figure 25 As shown in (A), the body weight of mice in the model group continued to decrease, while the mice in the combined treatment group were more active compared to the model group, indicating that the mice recovered well and grew normally.

[0093] The wet weight to dry weight (W / D) ratio of lung tissue can reflect the degree of interstitial edema in the lungs. For example... Figure 25 As shown in (B), the W / D ratio of mice in the model group was significantly higher than that in the blank group, while the W / D ratio of mice in the combined treatment group of emodin and meropenem was significantly reduced, indicating that the combined treatment of emodin and meropenem can alleviate the degree of pulmonary edema.

[0094] In addition, 72 hours after infection, such as Figure 25 As shown in (C), the mice treated with the combination of emodin and meropenem had significantly lower bacterial loads in their lungs than the other treatment groups, indicating that the combination of emodin and meropenem showed good therapeutic effects on mice infected with Klebsiella pneumoniae.

[0095] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Use of emodin in the preparation of drugs against meropenem-resistant Klebsiella pneumoniae.

2. The use according to claim 1, characterized in that, The meropenem-resistant Klebsiella pneumoniae includes ATCC® BAA1705™.

3. The use according to claim 1 or 2, characterized in that, The emodin enhances the bactericidal effect of antibiotics by disrupting the formation of biofilms in meropenem-resistant Klebsiella pneumoniae.

4. The use according to claim 1 or 2, characterized in that, The emodin enhances the bactericidal effect of the antibiotic by binding to KPC-2 carbapenemase produced by meropenem-resistant Klebsiella pneumoniae and inhibiting the activity of KPC-2 in hydrolyzing meropenem. And / or, the emodin enhances the bactericidal effect of the antibiotic by altering the functional group composition and secondary structure of the KPC-2 protein, thereby inhibiting the activity of KPC-2 in hydrolyzing meropenem. And / or, the key binding sites of the emodin to the KPC-2 protein are TRP-105 and THR-237.

5. The use of an antibacterial agent in the preparation of a drug for treating pneumonia caused by KPC-2 positive bacteria; wherein the bacteria is Klebsiella pneumoniae; the active ingredients of the antibacterial agent include emodin and meropenem antibiotics; and the dosages of emodin and meropenem antibiotics in the antibacterial agent are 12.5~25 mg / kg and 5 mg / kg, respectively.

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