CN115645414B - Antibacterial pharmaceutical composition and application thereof - Google Patents

Abstract

The invention provides an antibacterial pharmaceutical composition and its application. The present invention examines the antibacterial and anti-biofilm activities of triptolide against enterococci, as well as its ability to assist in restoring the sensitivity of VRE to vancomycin. Research results show that triptolide has significant antibacterial and anti-biofilm activities against enterococci, including VRE. Subminimum inhibitory concentrations of triptolide restored vancomycin activity against VRE in vitro and in vivo. The combination of vancomycin and triptolide has a synergistic effect, and triptolide is expected to be used as a new antibacterial agent or a sensitizer for vancomycin, providing an option for the treatment of VRE.

Description

Antibacterial pharmaceutical compositions and their applications

Technical field

The present invention relates to the field of biomedicine, specifically, to an antibacterial pharmaceutical composition and its application.

Background technique

Enterococci are commensal bacteria that mainly exist in the gastrointestinal tract of humans and animals and are considered to be one of the main causes of nosocomial infections (Agudelo Higuita et al., 2014; Kim et al., 2019). More than 20 types of enterococci have been identified, of which Enterococcus faecalis and Enterococcus faecium are the most clinically relevant species. The most common infections caused by them are urinary tract infection, bacteremia, Endocarditis and surgical site infection. The mortality rate of severe enterococcal infections is as high as 20%-40% (Miro et al., 2013). Studies have shown that nosocomial infections with ampicillin- and vancomycin-resistant enterococci have been on the rise over the past decade (Haque et al., 2018). The emergence and spread of multidrug-resistant enterococci, especially vancomycin-resistant enterococci (VRE), pose a serious threat to clinical treatment. The World Health Organization includes vancomycin-resistant Enterococcus faecium on its list of “high priority pathogens” (WHO, 2017). Therefore, there is an urgent need to develop new antibacterial agents with new skeletons or discover new strategies to treat infections caused by VRE.

Natural products have provided people with a rich source of drugs since ancient times. Active compounds such as ipecahine, artemisinin, houttuynia cordata, and berberine are all representatives of plant-derived anti-infective drug lead compounds. The core structure and active groups of monomeric compounds isolated from natural products are formed through long-term natural selection. The biological activities displayed during the screening process have advantages that cannot be matched by artificially synthesized compounds. Therefore, natural products with diverse structures and their derivatives play an extremely important role in the research of innovative drugs.

Celastrol (CEL) is the main bioactive component of Tripterygium wilfordii and has attracted widespread attention for its multiple promising bioactivities, including anti-cancer, anti-inflammatory, weight loss, cardioprotection, antibacterial, antioxidant, and anti-inflammatory properties. Allergy, neuroprotection, anti-thrombosis, anti-osteoarthritis and anti-Alzheimer's disease, etc. (Hou et al., 2020). It has been reported that triptolide has antiplankton and anti-biofilm activities against Staphylococcus aureus (Ooi et al., 2015; Woo et al., 2017). There are no relevant research reports on the anti-enterococci activity of triptolide.

Contents of the invention

The purpose of the present invention is to provide a novel antibacterial pharmaceutical composition and its application.

In order to achieve the object of the present invention, in the first aspect, the present invention provides the use of triptolide in preparing antibacterial drugs or compositions; wherein the bacteria are enterococci and drug-resistant bacteria thereof.

In the present invention, the enterococci are preferably Enterococcus faecalis (E.faecalis) and Enterococcus faecium (E.faecium), as well as their drug-resistant bacteria.

Further, the drug-resistant bacteria may be selected from vancomycin-resistant enterococci (VRE) and ampicillin-resistant enterococci, such as vancomycin-resistant Enterococcus faecalis and vancomycin-resistant Enterococcus faecium.

In a second aspect, the present invention provides the use of triptolide in preparing FtsZ protein inhibitors.

Preferably, the FtsZ protein is derived from enterococci and drug-resistant bacteria.

In a third aspect, the present invention provides a new antibacterial pharmaceutical composition, the active ingredients of which are triptolide and vancomycin.

Further, the mass ratio of tripterine to vancomycin is 1:16 to 1:2.

In a fourth aspect, the present invention provides the use of the composition in preparing antibacterial biological products; wherein the bacteria are enterococci and drug-resistant bacteria thereof.

Furthermore, triptolide and vancomycin can be administered separately in any order, or triptolide and vancomycin can be administered simultaneously.

Through the above technical solutions, the present invention has at least the following advantages and beneficial effects:

Compared with the prior art, the present invention at least has the following advantages:

The present invention examines the antibacterial and anti-biofilm activities of triptolide against enterococci, as well as its ability to assist in restoring the sensitivity of VRE to vancomycin. Research results show that triptolide has significant antibacterial and anti-biofilm activities against enterococci, including VRE. In vitro and in vivo, sub-MIC concentrations (subminimum inhibitory concentration) of triptolide restored the activity of vancomycin against VRE. The combination of vancomycin and triptolide has a synergistic effect, and triptolide is expected to be used as a new antibacterial agent or a sensitizer for vancomycin, providing an option for the treatment of VRE.

Description of the drawings

Figure 1 shows the activity of different concentrations of CEL against Enterococcus strains in the bactericidal curve method in the preferred embodiment of the present invention. A: Enterococcus faecalis ATCC 700802 (MIC=4μg/mL); B: Enterococcus faecalis ATCC 51575 (MIC=2μg/mL); C: Enterococcus faecium ATCC 700221 (MIC=2μg/mL); D: Enterococcus faecium ATCC 51559 (MIC=2μg/mL).

Figure 2 shows the concentration-dependent activity of CEL, vancomycin and ampicillin in clearing biofilms in the preferred embodiment of the present invention (n=3, ***P<0.001).

Figure 3 shows the activity of sub-MIC concentrations of CEL and vancomycin used alone and in combination against VRE strains in the preferred embodiment of the present invention. A: Enterococcus faecalis ATCC 700802, 1/8MIC (0.5μg/ml) CEL and <1/128MIC (2μg/ml) vancomycin (CELMIC=4mg/mL, vancomycin MIC=16μg/mL); B : Enterococcus faecium ATCC 700221, 1/4MIC CEL (0.5μg/ml) and <1/64MIC (16μg/ml) vancomycin (MIC of CEL=2mg/mL, MIC of vancomycin>256μg/mL).

Figure 4 shows the protective effect of CEL and vancomycin alone or in combination against lethal enterococcal infection of Galleria mellonella larvae in the preferred embodiment of the present invention. A: Enterococcus faecalis ATCC 700802; B: Enterococcus faecium ATCC 700221. There are 10 larvae in each group. The infectious dose is 2×10 ⁶ CFU/animal.

Figure 5 is a scanning electron microscope photograph of Enterococcus faecalis ATCC 700802 and Bacillus subtilis ATCC 21332 without drug treatment and after CEL treatment for 4 hours in the preferred embodiment of the present invention. A1: Enterococcus faecalis ATCC 700802, control, magnification 5000 times; A2: Enterococcus faecalis ATCC 700802, control, magnification 20000 times; B1: Enterococcus faecalis ATCC 700802, 2 μg/mL CEL treatment, magnification 5000 times; B2: Enterococcus faecium Cocci ATCC 700802, 2 μg/mL CEL treatment, amplification 20,000 times; C: Bacillus subtilis ATCC 21332, control; D: Bacillus subtilis ATCC 21332 2 μg/mL CEL treatment.

Figure 6 shows the docking results of CEL and FtsZ proteins in the preferred embodiment of the present invention, including the interaction of different amino acids between CEL and FtsZ.

Figure 7 shows the surface plasmon resonance detection results obtained on the FtsZ coated chip under different concentrations of CEL in the preferred embodiment of the present invention.

Figure 8 is a dose-response curve of CEL inhibiting the GTPase activity of Enterococcus faecalis FtsZ in a preferred embodiment of the present invention. Each point represents three independent experiments.

Detailed ways

The present invention aims to study the antibacterial and anti-biofilm activities of triptolide against enterococci and its ability to assist in restoring the sensitivity of VRE to vancomycin.

Minimum inhibitory concentration (MIC) determination, biofilm clearance experiment and time-killing curve experiment were used to study the in vitro antibacterial activity of triptolide. The synergy between triptolide and vancomycin was determined using the checkerboard method and the bactericidal curve method. In vivo studies were performed on a Galleria mellonella larvae infection model. The potential antibacterial mechanism of triptolide was explored through molecular docking, biomolecule binding interactions, and the enzyme inhibitory effect of triptolide on bacterial division protein FtsZ.

The results of the study showed that triptolide inhibited all tested Enterococcus faecalis and Enterococcus faecium strains, with an MIC range of 0.5 to 4 μg/mL. In the bactericidal curve analysis, triptolide inhibited bacterial growth in a concentration-dependent manner. After 24 hours of exposure at a concentration of 16 μg/mL, triptolide can remove more than 50% of the biofilm. The combination of triptolide and vancomycin showed synergistic effects against all 23 tested strains, with a median fractional inhibitory concentration index (FICI) of 0.25 in the checkerboard experiment. The combined use of sub-MIC levels of triptolide and vancomycin showed synergistic effects in bactericidal curve experiments and significant protective effects in a Galleria mellonella larvae infection model compared to either drug alone. Tripterine has strong binding and inhibitory abilities to FtsZ, with Kd and IC ₅₀ of 1.75±0.06μM and 1.04±0.17μg/mL respectively.

The following examples are used to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available commodities.

The abbreviations and terms used in this invention are as follows:

VRE: vancomycin-resistant enterococci;

CEL: triptolide;

MIC: minimum inhibitory concentration;

CFU: colony forming unit;

FICI: fractional inhibitory concentration index;

VSE: vancomycin-susceptible enterococci;

MHA: Mueller-Hinton agar;

CAMHB: cation-adjusted Mueller-Hinton broth;

BHI: brain heart infusion;

PBS: phosphate buffered saline;

SD: standard deviation;

CLSI: Clinical and Laboratory Standards Institute;

SEM: scanning electron microscope;

SPR: Surface Plasmon Resonance.

Example 1 Study on antibacterial activity of triptolide

1. Materials and methods

1.1 Strains and growth conditions

All strains used in the present invention are from the Center for the Collection of Pathogen Microorganisms and Viruses of the Chinese Academy of Medical Sciences (CAMS-CCPM-A), including 5 ATCC standard VRE strains (2 strains of Enterococcus faecium and 3 strains of Enterococcus faecalis), 18 strains Clinical VRE strains (17 Enterococcus faecium and 1 Enterococcus faecalis), 5 ATCC standard vancomycin-susceptible enterococci (VSE) strains (2 Enterococcus faecium and 3 Enterococcus faecalis), 72 clinical isolates of VSE strains (31 Enterococcus faecalis and 41 Enterococcus faecalis). Strains are typically grown in cation-conditioned Mueller-Hinton broth (CAMHB) or Mueller-Hinton agar (MHA) at 37°C.

1.2 Chemicals and Reagents

Vancomycin, ampicillin, and triptolide were purchased from China Institute of Pharmaceutical and Biological Products (Beijing, China). Dissolve vancomycin, triptolide and ampicillin in distilled water, dimethyl sulfoxide and phosphate buffer (pH 8.0) respectively as a stock solution, with a concentration of 10 mg/mL, and filter with a 0.22 μm filter Store at -20°C. CAMHB, MHA and brain heart infusion (BHI) used for bacterial culture and antimicrobial susceptibility experiments were purchased from BD (Franklin Lakes, NJ, USA).

CytoPhos phosphate assay Biochem kit and recombinant Enterococcus faecalis FtsZ protein (FTZ04-B) were purchased from Cytoskeleton (Denver, Colorado, USA).

1.3 Sensitivity experiment

All strains were stored at -80°C, streaked on MHA plates and cultured overnight before testing. The minimum inhibitory concentrations (MIC) of triptolide and vancomycin were determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2021), with ampicillin as the quality control antibiotic. Compounds were added to the wells of a 96-well microtiter plate at a starting concentration of 256 μg/mL and serially diluted in CAMHB medium. Add 10 μL of bacterial suspension to make the final inoculum volume reach 5 × 10 ⁵ CFU/mL, incubate the plate at 37°C for 24 hours, and then read the MIC. The MIC is defined as the lowest concentration of a compound that inhibits bacterial growth. The vancomycin MIC breakpoints are as follows: ≤4 μg/mL, sensitive; 8-16 μg/mL, intermediate; ≥32 μg/mL, resistant.

1.4 Time bactericidal curve of triptolide

The kinetics of triptolide's bactericidal activity was evaluated by bactericidal curve experiments according to the method described by CLSI (CLSI, 1999). The experiments were conducted on Enterococcus faecalis ATCC 700802, ATCC 51575, Enterococcus faecalis ATCC700221, and ATCC51559. Briefly, the bacterial solution cultured overnight was diluted with CAMHB to a final concentration of approximately 2 × 10 ⁶ CFU/mL, and aliquoted into sterile glass tubes, 10 mL per tube. Different concentrations of tripterine (1/4MIC, 1/2MIC, MIC, 2MIC and 4MIC) containing bacteria but not tripterine were obtained by adding a volume of tripterine stock solution to each test tube. test tubes were used as growth controls. The bacterial count of the bacterial solution was determined at 0, 2, 4, 6, 8 and 24 hours of incubation. After 10-fold serial dilution of the bacterial solution, 10 μL samples were dripped on the MHA plate in triplicate, and after incubation at 37°C for 24 hours, viable bacteria were counted and recorded as log ₁₀ CFU/mL. Bactericidal activity was defined as a reduction in colony count of ≥3 log ₁₀ CFU/mL (99.9% clearance) compared to the starting inoculum. The detection limit is 2log ₁₀ CFU/mL.

1.5 Biofilm removal experiment

Biofilm clearance experiments were performed on the study strains as previously described (Wang et al., 2019). Enterococcus faecalis ATCC700802 was inoculated into BHI broth and shaken at 37°C overnight. After diluting the bacterial solution 1:100, add it to a 96-well plate treated with tissue culture (100 μL per well), and incubate at 37°C for 24 hours to form a biofilm. Gently remove the medium and add 100 μL of fresh medium containing varying concentrations of triptolide or antibiotic control (3 biological replicates). After culturing for an additional 24 hours, the medium was removed, the biofilm was gently washed three times with phosphate buffered saline (PBS), and heat-fixed at 60°C for 1 hour. Add 50 μL of 0.06% crystal violet to each well for staining for 5 min, and then wash repeatedly with distilled water to remove excess dye. Crystal violet was eluted from the stained biofilm by adding 200 μL of 30% acetic acid to each well and transferred to another ₉₆ -well plate for OD reading.

1.6 Statistical analysis

Statistical significance was determined using SPSS 16.0 one-way analysis of variance (ANOVA), and P values <0.05 were considered statistically significant.

2. Experimental results

Antibacterial activity of triptolide: The MIC values of triptolide and vancomycin against 52 strains of Enterococcus faecalis and 48 strains of Enterococcus faecium are shown in Table 1. 23% of the strains (23/100) were resistant to vancomycin , including 4 strains of Enterococcus faecalis and 19 strains of Enterococcus faecium. Triptolide shows antibacterial effects against all vancomycin-sensitive and resistant strains, with an MIC range of 0.5 to 4 μg/mL. This may indicate that triptolide exerts antibacterial activity through a mechanism different from vancomycin and is not easily Cross-resistance to vancomycin.

As shown in Figure 1, triptolide has a concentration-dependent antibacterial effect on enterococci. For all strains tested, 24 hours after inoculation, triptolide showed bactericidal effect on Enterococcus faecalis ATCC 700802, ATCC 51575, Enterococcus faecium ATCC 700221, and ATCC 51559 respectively at a concentration of 4MIC. Triptolide at a concentration of 2 MIC for each strain showed antibacterial activity against the four strains and lasted until 24 hours. Regarding the MIC concentration, Enterococcus faecalis ATCC 700802 was still inhibited by triptolide after 24 hours of incubation, whereas bacterial growth was observed in the other three strains. Triptolide at 1/4 and 1/2 MIC showed no obvious antibacterial activity against all strains.

Biofilms have been associated with refractory infections such as endocarditis, osteomyelitis, chronic wound infections, catheter-related infections, and prosthetic joint infections, and are also used as models for studying drug resistance. The present invention further evaluates the effect of triptolide on VRE biofilm. As commonly used antibiotics in the clinical treatment of enterococcal infections, ampicillin or vancomycin have little effect on the removal of biofilms. In comparison, 16 μg/mL of triptolide cleared more than 50% of the E. faecalis ATCC 700802 biofilm after 24 hours of exposure (Figure 2).

Table 1 Minimum inhibitory concentrations of triptolide and vancomycin against 100 enterococcal standard strains and clinical isolates

Example 2 Synergistic effect of triptolide and vancomycin in vitro

1. Experimental methods

The synergistic effect of triptolide and vancomycin was evaluated on 23 VRE strains (including 5 standard strains and 18 randomly selected clinical isolates) using the microdilution checkerboard method (Wang et al., 2019). Briefly, the bacterial solution was added to the wells of a 96-well plate containing serial dilutions of triptolide and vancomycin, and the bacterial amount was 5 × 10 ⁵ CFU/mL. The dilution ranges of triptolide and vancomycin used in the checkerboard method were 0.125 to 8 μg/mL and 0.25 to 256 μg/mL, respectively. After 24 hours of incubation at 37°C, the fractional inhibitory concentration index (FICI) was calculated using the following equation to analyze the combined effect of triptolide and vancomycin. The experiment was repeated three times:

FICI=(MIC of drug A in combination/MIC of drug A alone)+(MIC of drug B in combination/MIC of drug B alone)

When FICI is ≤0.5, it is synergistic; when 0.5≤FICI≤4, it is irrelevant; when FICI>4, it is antagonistic.

Bactericidal curve experiments were conducted on Enterococcus faecalis ATCC 700802 and Enterococcus faecium ATCC 700221 to evaluate the bactericidal kinetics of triptolide and vancomycin. The time bactericidal curve study of combination therapy is similar to that of triptolide alone. A drug-free bacterial solution was included for each strain as a growth control. Triptolide and vancomycin were tested at sub-MIC concentrations of each drug. Colony counts were determined at 0, 2, 4, 6, 8 and 24 hours. The synergistic effect is defined as the reduction of 24-hour viable bacterial count in the combination group by ≥2log ₁₀ CFU/mL compared with the more active single-use group, and at the same time, the 24-hour viable bacterial count in the combined group is compared with the initial inoculation amount. Decrease ≥2log ₁₀ CFU/mL.

2. Experimental results

The results of the chessboard method experiment are shown in Table 2. When vancomycin was combined with triptolide of 1/8-1/4 MIC (0.25-1 μg/mL), the inhibitory concentration of vancomycin on the strain was significantly reduced to its 1/128-1/4 MIC (2- 16μg/mL). According to the FIC index, triptolide showed a synergistic effect on 23 strains of enterococci when combined with vancomycin.

The results of the bactericidal curve when triptolide and vancomycin are combined are shown in Figure 3. Vancomycin alone has poor inhibitory activity against bacterial growth at sub-MIC concentrations. The inhibitory effect of triptolide alone on bacterial growth was weak, and bacterial regeneration was observed 6 to 24 hours after inoculation. Over 24 hours, CFU counts for vancomycin and triptolide alone were identical to growth control levels for the respective strains. In contrast, the combination of triptolide and vancomycin showed a strong synergistic effect on both tested strains. After 24 hours of culture, compared with the use of triptolide and vancomycin alone, the combined use can significantly reduce the number of viable bacteria by more than 3log10 CFU/mL. After 24 hours, the inhibitory effect on the growth of all strains was still observed. In addition, compared with the initial inoculum, the CEL-vancomycin combination reduced the number of viable bacteria by 2log10CFU/mL.

Table 2 The results of the checkerboard method of triptolide combined with vancomycin on 23 strains of enterococci

Example 3 Synergistic effect of triptolide and vancomycin in vivo

1. Establishment of Galleria mellonella larvae infection model

Galleria mellonella larvae were left at room temperature to acclimate for 24 hours. Larvae with a weight between 270 and 330 mg and a size of about 2 cm were selected for the test. Enterococcus faecalis ATCC 700802 and Enterococcus faecium ATCC 700221 were streaked on BHI agar plates and cultured overnight in BHI. The overnight cultures of the two strains were washed with PBS and adjusted to an appropriate density (approximately 2×10 ⁸ CFU/mL), and 10 μL of bacterial solution was injected into the right hind foot of the larvae using a glass microsyringe. One hour after infection, the larvae were injected with vancomycin, triptolide, or a combination of vancomycin and triptolide through the left foot, with 10 larvae in each group. In addition, 10 larvae were injected with 10 μL PBS as a negative control. Larvae were cultured at 35°C and monitored continuously for 96 h after infection. When the larvae did not respond to mechanical stimulation, they were determined to be dead, and the survival curves of each group were drawn.

Multiple comparison analysis of survival data was performed by log-rank test combined with Bonferroni correction using Kaplan-Meier survival analysis with GraphPad Prism 8.

2. Result

The CEL-vancomycin combination showed a protective effect on Galleria mellonella larvae against lethal infectious doses of VRE strains Enterococcus faecalis ATCC 700802 and Enterococcus faecium ATCC 700221. After infecting the larvae with 2×10 ⁶ CFU of bacteria, 10 μl PBS, 0.25 mg/kg CEL, 40 mg/kg vancomycin or CEL-vancomycin combination was injected. All larvae treated with PBS, CEL or vancomycin alone died within 48 hours after inoculation with VRE. CEL or vancomycin alone were not sufficient to provide protection, while 80% of larvae infected with Enterococcus faecalis ATCC 700802 and 90% of larvae infected with Enterococcus faecium ATCC 700221 survived for more than 96 hours after combined treatment. survival time (Figure 4). Therefore, the combined use of CEL and vancomycin has an effective protective effect against fatal VRE infection.

Example 4 Research on Cellular Action Mechanism

1. Scanning electron microscope (SEM)

Overnight cultures of Enterococcus faecalis ATCC 700802 and Bacillus subtilis ATCC 21332 were diluted 1:100 in CAMH broth and grown at 37°C to logarithmic growth phase (OD _{600 nm} = 0.4), and the bacteria were then incubated with or without The cells were cultured in a medium containing sub-MIC level CEL for 4 hours, washed with PBS, fixed with 2.5% glutaraldehyde for 24 hours, dehydrated through an ethanol gradient, dried, and sprayed with gold, and then observed under a scanning electron microscope (Hitachi SU8020, Japan).

2. Docking analysis

Discovery Studio 4.5 software was used to conduct small molecule docking studies using the 3D structure (PDB number: 5MN4) of FtsZ protein (ID: 60893384 in NCBI). Proteins are normalized to determine important amino acids in the predicted binding pocket. After energy minimization, Libdock was used to dock all conformations of CEL with the sites of the selected active cavity. Docking compounds are scored based on how they bind at the binding site. 3. Surface plasmon resonance (SPR) analysis

SPR analysis was performed on a BIAcore T200 biosensor system (GE Healthcare Life Sciences, Piscataway, NJ, USA) at 25°C using a CM5 chip. Different concentrations of CEL (25, 12.5, 6.25, 3.1, 1.6 μM) were combined with FtsZ in 1×PBS-P+ (GE Healthcare LifeSciences) at a flow rate of 20 μL/min for 120 s. After each binding reaction, a 60 s dissociation time was used to allow the signal to return to baseline. Kd values were calculated using BiacoreT200 evaluation software (GE Healthcare Life Sciences version 2.0, Piscataway, NJ, USA). 4. Determination of GTPase activity

The GTPase activity of the recombinant Enterococcus faecalis FtsZ protein was determined using the CytoPhos phosphate detection biochemical kit (Cytoskeleton, USA) (Sun et al., 2017) as described in the literature. Enterococcus faecalis FtsZ protein (0.5 μM) was mixed with solvent (1% dimethyl sulfoxide) or different concentrations of CEL (0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32 μg/mL) in Incubate in 20mM Tris buffer (pH7.5) for 10 minutes at room temperature. Then add _5mM MgCl and 200mM KCl. Start the reaction by adding 500 μM GTP and react at 37°C. After 30 minutes, stop the reaction by adding 140 μL of Cytophos reagent and incubate for 10 minutes. Inorganic phosphates were quantified by measuring absorbance values at 650 nm using a microplate reader (BioRad Laboratories Ltd., UK). Relative _IC50 values were determined by nonlinear regression using a sigmoidal concentration response curve in GraphPad Prism 9 (GraphPad Software, La Jolla, CA). All experiments were analyzed three times independently.

5. Experimental results

Changes in bacterial cell morphology can often provide valuable clues to the mechanism of antibacterial action and are often used in preliminary mechanism studies. Through scanning electron microscopy observation of bacterial cell morphology, the mechanism of CEL's antibacterial effect was deeply explored for the first time. As shown in Figure 5, treatment with CEL did not cause any observable bacterial surface abnormalities compared to untreated cells. However, in the CEL-treated group, bacteria were observed to become significantly longer and "candied haws-shaped", indicating that bacterial division was inhibited. To further confirm our hypothesis, we performed cell morphology studies with Bacillus subtilis. The length of B. subtilis increased significantly after the addition of CEL compared with untreated cells. From this, we speculate that CEL displays its antibacterial activity by inhibiting bacterial division.

Due to its important role and high conservation in all bacteria, filamentous temperature-sensitive mutant Z (FtsZ) has become a target of great concern for new antibacterial drugs (Schaffner Barbero et al., 2012). FtsZ aggregates into protofilaments through GTP-dependent polymerization, and further self-assembles into the Z ring, a key organelle for bacterial cell division, recruiting other proteins to jointly drive bacterial division and the formation of new cell poles.

Based on the findings under scanning electron microscopy (SEM) and the important role of FtsZ protein in bacterial cell division, we proposed the hypothesis that CEL may be antibacterial by inhibiting the function of FtsZ and its related biological activities. We predict that FtsZ may be one of the targets of CEL's antibacterial effects.

This invention carried out molecular docking between CEL and FtsZ for the first time. Discovery Studio 4.5 software was used for molecular docking. As shown in Figure 6, CEL can fit the active pocket of FtsZ protein well, with a docking score of 113.1. Predicted interactions between small molecules and proteins include two hydrogen bonds between the oxygen atom on the x-carbonyl or x-carboxyl group and ARG143 or THR133 residues, and one Pi anion bond between ring A and GLU139. Together, these interactions promote binding, suggesting that the FtsZ protein may be a direct target of CEL.

To further confirm the interaction between CEL and FtsZ, the binding between the two was analyzed by SPR. The results showed that CEL could bind to FtsZ in a dose-dependent manner, with a Kd value of 2.454 μM (Figure 7), indicating its strong binding ability to FtsZ.

Since the assembly kinetics of FtsZ is thought to be regulated by its GTPase activity, the inhibitory effect of CEL on the GTPase activity of E. faecalis FtsZ was evaluated. CEL was found to significantly inhibit the GTPase activity of Enterococcus faecalis FtsZ protein, with an IC ₅₀ value of 1.04±0.17 μg/mL (Figure 8). There is a good correlation between GTPase inhibitory activity and antibacterial activity, indicating that the compound interferes with bacterial growth through binding to FtsZ and inhibiting GTPase function. The antibacterial mechanism of CEL that is different from antibiotics also explains its synergistic effect with vancomycin.

In summary, CEL has antibacterial and antibiofilm activity against enterococci, including VRE strains, and restores the activity of vancomycin against VRE in vitro and in vivo. Overall, CEL is expected to become a new antibacterial agent and antibacterial adjuvant, providing a new treatment option against VRE.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

references:

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Claims (1)

1. the application of the antibacterial pharmaceutical composition in preparing antibacterial biological products; wherein the bacteria are selected from vancomycin-resistant enterococcus faecium and vancomycin-resistant enterococcus faecium;

the active ingredients of the antibacterial pharmaceutical composition are tripterine and vancomycin;

the mass ratio of the tripterine to the vancomycin is 1:16-1:2.