CN117100837A - Pharmaceutical composition for resisting drug-resistant enterococcus and application thereof - Google Patents

Abstract

The invention relates to the field of biological medicine, and provides a medicine composition for resisting drug-resistant enterococcus and application thereof. The active ingredients of the drug-resistant enterococcus pharmaceutical composition are gambogic acid and vancomycin. The invention discloses for the first time that gambogic acid has good antibacterial activity on enterococci including vancomycin-resistant enterococci (VRE) strains, and can restore the activity of vancomycin on VRE in vitro and in vivo. Gambogic acid is likely to become an antibacterial sensitizer of vancomycin, and provides a new therapeutic choice for VRE resistance.

Description

Pharmaceutical composition for resisting drug-resistant enterococcus and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a medicine composition for resisting drug-resistant enterococcus and application thereof.

Background

Enterococci are an important component of the intestinal microbiota of healthy humans and animals. Such opportunistic pathogens cause infectious diseases such as urinary tract infections, surgical site infections, bacteremia and endocarditis, especially in immunocompromised patients. As one of the main causes of hospital-acquired infection (HAI), 2011 to 2014Enterococci make up 14% of HAI in the united states; between 2010 and 2020, 10.9% of HAI is found in the european community of the world health organization. The most common enterococcus in clinical infection is enterococcus faecalis @Enterococcus faecalis,E. faecalis) Enterococcus faeciumEnterococcus faecium, E. faecium). In recent years, the treatment of enterococcus infections has become more challenging due to the emergence of drug resistant strains. Drug resistant strains can develop resistance to a variety of antibiotics, including Vancomycin (VAN), the last line of defense against gram-positive bacterial infection. Vancomycin-resistant enterococci (VRE) are widely spread worldwide, with up to 80% of enterococci faecium being resistant to VAN in some areas. Vancomycin-resistant enterococcus faecium is listed by the world health organization as a "highly preferential pathogen". The emergence and spread of vancomycin-resistant enterococci has a tremendous impact on public health and economy. Thus, there is an urgent need to develop new antibacterial agents or new strategies with unique structures to combat VRE infection. Given the long development cycle of new chemical entity drugs, a high degree of dislocation between clinical needs and drug innovations, combination antimicrobial treatment of drug-resistant bacterial infections is often recommended. Thus, the search for new antimicrobial sensitizers is a powerful strategy to bring first-line antibiotics back to the antimicrobial battlefield.

Natural product monomers have a wide range of pharmacological activities, especially in the anti-tumor and anti-infective fields. Gambogic Acid (GA) is a natural xanthone compound extracted from dry resin of gamboge of gambogaceae, and has various biological activities of enhancing immunity, resisting inflammation, resisting tumor, resisting oxidation, resisting bacteria, resisting virus, etc., and its structure is shown in figure 1. GA is reported to exhibit antibacterial activity against methicillin-resistant staphylococcus aureus (MRSA) and also to inhibit its biofilm formation and intracellular infection of bacteria. However, the synergistic effect of GA in combination with VAN, especially on VRE, is not reported.

Disclosure of Invention

The invention aims to provide a drug combination for resisting drug-resistant enterococcus and application thereof.

The invention provides a promising synergistic antibacterial combination of VAN and GA. The present invention systematically evaluates the antibacterial activity of GA against enterococcus faecalis and enterococcus faecium, alone and in combination with VAN, particularly against VRE in vitro and in a mouse multiple organ infection model. The effect of GA on type II DNA topoisomerase (including its specific subunits) was also investigated to investigate its potential antibacterial mechanism of action as an anti-enterococcus drug.

In order to achieve the aim of the invention, in a first aspect, the invention provides a drug combination for resisting drug-resistant enterococci, and active ingredients of the drug combination are gambogic acid and vancomycin.

Further, the mass ratio of gambogic acid to vancomycin in the pharmaceutical composition is 1:2-1:32.

In a second aspect, the invention provides the use of said pharmaceutical composition for the preparation of a biologic against drug-resistant enterococci.

The drug-resistant enterococci comprise vancomycin-resistant enterococci and vancomycin-resistant enterococcus faecium.

Further, gambogic acid and vancomycin are administered separately in no-order or simultaneously together.

According to the invention, the in-vitro antibacterial activity of the GA and the VAN combination on the VAN sensitive strain and the drug-resistant strain is examined, and in-vivo activity research is carried out on a mouse multi-organ infection model, and meanwhile, the potential antibacterial mechanism of the GA is discussed. The results showed that GA showed potent in vitro antibacterial activity against all tested strains with minimum inhibitory concentration (minimal inhibitory concentration, MIC) ranging from 2-4. Mu.g/mL. The combination of GA and VAN showed synergistic antibacterial effects on 18 of the 23 VRE strains tested; in the bactericidal curve test, a synergistic antibacterial effect was exhibited on 4 test strains. In a mouse multiple organ infection experiment, combination treatment showed a significant reduction in tissue bacterial load compared to either compound used alone. GA is able to bind to and inhibit the activity of the ParE subunit of topoisomerase IV, a bacterial type II DNA topoisomerase. The above results indicate that GA has significant antibacterial activity against enterococci, and that GA at sub-MIC concentrations can restore antibacterial activity of VAN against VRE in vitro and in vivo. GA is expected to be a novel antibacterial potentiator of VAN for the treatment of VRE-induced infections.

Drawings

FIG. 1 shows the chemical structural formula of gambogic acid.

FIG. 2 is a graph showing the time sterilization profile of enterococci for 4 μg/mLVAN, 0.5 μg/mL GA, and a combination of both in a preferred embodiment of the invention. Among them, A, B, enterococcus faecalis ATCC 700802 and 09-9; C. d, enterococcus faecium ATCC 700221 and 12-1. And ∈ζ represents a growth control; represents 4. Mu.g/mL VAN; o represents 0.5. Mu.g/mL GA; represents 4. Mu.g/mL VAN+0.5. Mu.g/mL GA.

FIG. 3 shows the synergy of GA and VAN in a multiple organ infection model of mice in accordance with a preferred embodiment of the present invention.

FIG. 4 shows the morphology of enterococcus faecalis ATCC 700802 under a scanning electron microscope either untreated or GA treated in accordance with a preferred embodiment of the present invention. Wherein, A1, A2, GA treated bacteria; b1, B2, untreated bacteria.

FIG. 5 shows the effect of GA (A1, B1), neomycin (A2, B2) and ciprofloxacin (A3, B3) on bacterial gyrase and topoisomerase IV activity in a preferred embodiment of the invention.

FIG. 6 shows the effect of GA and neomycin on topoisomerase IV ATPase activity in a preferred embodiment of the invention. A, GA; b, neomycin.

FIG. 7 shows the interaction of GA with ParE subunit of enterococcus faecalis in a preferred embodiment of the invention. Wherein, A, different concentrations of GA and ParE are shown in a Surface Plasmon Resonance (SPR) sensorgram. B, inhibition of atpase activity of recombinant expressed enterococcus ParE by GA.

FIG. 8 shows the molecular docking of GA and the ParE subunit of enterococcus topoisomerase IV in a preferred embodiment of the present invention.

Detailed Description

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

Example 1 synergistic action of gambogic acid and vancomycin

The present invention aims to develop Gambogic Acid (GA) as one kind of antiseptic synergist to restore the sensitivity of VRE to VAN.

1. Materials and methods

1.1 Sensitivity test

allenterococcusstrainsusedinthepresentinventionwerefromtheCAMSpathogenicmicroorganismbacteriumandviruscollectioncenterpharmaceuticalmicroorganismcenter(CAMS-CCPM-A). According to Clinical and laboratory standards institute (Clinical)&Laboratory Standards Institute, CLSI), minimum inhibitory concentration MIC was determined using broth microdilution with Ampicillin (AMP) as a quality control drug. Compounds at an initial concentration of 1024. Mu.g/mL were serially double diluted in 96-well plates. 10 mu L of bacterial suspension was added to each well, the final concentration of bacteria was 5X 10 ⁵ CFU/mL, MIC was read after incubation at 37 ℃ for 24 hours. MIC is defined as the lowest drug concentration that inhibits bacterial growth.

1.2 Synergistic effect study

And (3) a microdilution chessboard method is adopted to examine the combined effect of GA and VAN combined medicines on 23 VRE strains. In both the lateral and longitudinal directions of a 96-well microtiter plate, GA and VAN were added in a final concentration of 5X 10 bacteria containing double serial dilutions of Mueller-Hinton broth (CAMHB) adjusted with cations ⁵ CFU/mL, after incubation at 37 ℃ for 24 hours, the effect of the combination was assessed using FIC index: fici= (MIC of drug a in combination/MIC of drug a alone) + (MIC of drug B in combination/MIC of drug B alone). FICI value less than or equal to 0.5 indicates synergistic activity, FICI less than or equal to 0.5 indicates additive effect, and FICI > 4 indicates antagonism.

Time sterilization curve determination was performed according to CLSI, and the overnight bacterial liquid was diluted to a final concentration of 2×10 ⁶ CFU/mL. GA or VAN or a combination of both was added to the sub MIC and colony counts were performed at 0, 2, 4, 6, 8 and 24 hours post inoculation. Each strain was provided with a growth control without any compound. In the sterilization curve experiment, the colony count is reduced by more than or equal to 2log when the combination group is compared with the single group with the best activity ₁₀ It is determined that there is a synergistic effect.

1.3 Mouse multi-organ infection model

CD-1 mice (18-20 g, male and female halves) were purchased from Beijing Bei Fu Biotechnology Co., ltd and used for study after one day of adaptation in animal barrier facilities. To induce systemic infection, mice were intraperitoneally injected with 1.0X10 ⁷ CFU/murine enterococcus faecalis ATCC 700802. Mice were randomly divided into four groups (10 mice per group) and injected twice with GA (5 mg/kg), VAN (5 mg/kg), GA+VAN (5 mg/kg+5 mg/kg) or solvent control (10% dextrose solution containing 10% ethanol and 10% Crenhor EL) at the tail vein 2 and 8 hours after infection, respectively. 24 hours after infection, mice were sacrificed and hearts, lungs, kidneys and livers were aseptically collected for homogenization and viable counts.

1.4 Scanning Electron Microscope (SEM)

Treatment of bacteria in log phase with GA at 1/2 MIC (2. Mu.g/mL) (OD ₆₀₀ =0.4) for 24 hours and fixed with PBS solution containing 2.5% glutaraldehyde for 24 hours. The mixture was dehydrated by ethanol gradient, dried, and sprayed with gold, and observed under a scanning electron microscope (Hitachi SU8020, japan).

1.5 Topoisomerase activity assay based on agarose gel

Staphylococcus aureus gyrase and topoisomerase IV holoenzymes were purchased from instralis and enzyme activity inhibition studies of GA were performed according to the manufacturer's instructions. Briefly, the enzyme was incubated with 0.5. Mu.g of plasmid pBR322 in its relaxed or supercoiled state and the desired concentration of compound, respectively, for 30 minutes at 37℃in a total volume of 30. Mu.L. The reaction was stopped by adding reaction stop buffer and chloroform/isoamyl alcohol (v: v, 24:1). Samples were loaded onto a 1% agarose gel and run at 75V for 2 hours, after staining with nucleic acid dye, the bands were photographed under an ultraviolet lamp.

1.6 ATPase assay of topoisomerase IV holoenzyme or subunit

The final concentration of 8.5nM of enzyme (holoenzyme or parE subunit) was added to a buffer of 40mM Tris (pH 7.5), 80mM sodium chloride, 8mM magnesium acetate and 1mM EDTA, 10. Mu.L per well volume, 30. Mu.L of ATP (4 mM) was added to initiate the reaction, incubated at 20℃for 30 min, 200. Mu.L was addedThe enzyme reaction was terminated by the solution of L malachite green. After 10 minutes the optical density at 620nm was measured with an enzyme-labeled instrument (BioTek Synergy 2) and the inorganic phosphate released during conversion of ATP to ADP was detected. Calculation of IC using GraphPad Prism 8 ₅₀ Values.

1.7 Expression and purification of proteins

Amplification of genomic DNA from enterococcus faecalis using In Fusion Snap Assembly Kit (TaKaRa)parEFull length was inserted into plasmid pET 28. The ligation vector was transformed into E.coli Rosetta (DE 3). Bacteria were cultivated to OD at 37℃in 50. Mu.g/mL kanamycin-resistant LB medium ₆₀₀ Up to 0.6-0.8 and protein expression was induced with 0.5mM IPTG at 30℃for 18 hours. After ultrasonic lysis, the supernatant was loaded onto a pre-equilibrated Ni-NTA column (GE Healthcare), 10 column volumes were washed with 25mM imidazole binding buffer, and gradient elution of 20 column volumes was performed using 10-250 mM imidazole. The protein was further purified on a size exclusion chromatography Superdex-200 column with 25mM Tris-HCl, pH7.5, 150mM NaCl solution.

1.8 Molecular docking and affinity analysis

Automatic docking studies were performed on the 3D structure of the ParE subunit of enterococcus faecalis V583 (PDB ID:4HZ 5) and GA using Discovery Studio 4.5. Libdock is used to interactively dock all conformational bodies of GA to selected active sites after energy minimization.

Surface Plasmon Resonance (SPR) assays were performed using a BIAcore T200 biosensor system (GE Healthcare Life Sciences, piscataway, NJ, USA) and CM5 chip at a temperature of 25 ℃. The assay measures the binding of ParE to different concentrations of GA at a flow rate of 20. Mu.L/min over a period of 120 s, and is used to calculate the Kd value.

1.9 Statistical analysis

Statistical analysis was performed by single factor analysis of variance using GraphPad Prism 8. P values <0.05 were considered statistically significant.

2. Results and discussion

2.1 Antibacterial Activity of GA

Table 1 summarizes the MIC of GA, VAN and ampicillin for 52 strains of enterococcus faecalis and 48 strains of enterococcus faecium. 23% (23/100) strains, including 4 enterococcus faecalis and 19 enterococcus faecalis, were identified as resistant to VAN. GA shows similar antibacterial effect on VAN sensitive strain and drug resistant strain, and MIC is 2-4 μg/mL.

Table 1 GA and VAN MIC (μg/mL) for enterococcus Standard strain and clinical isolate

Note that: a. the vanmid folding points of (c) are as follows: sensitive, less than or equal to 4 mug/mL; intermediating 8-16 mug/mL; drug resistance is more than or equal to 32 mug/mL. b. VAN, vancomycin. C. AMP, ampicillin.

2.2 Synergistic effects of GA and VAN

The results of the chessboard method are shown in Table 2. The combination of GA and VAN showed synergy for 18 strains in the 23 enterococci tested, with FIC index between 0.078 and 0.5. Among these 18 strains, when used in combination with GA at a concentration of 1/16 to 1/4 MIC (0.25-1. Mu.g/mL), the inhibitory concentration of VAN on the strain was significantly reduced to 1/1024 to 1/4 MIC (1-16. Mu.g/mL).

Table 2 chessboard method to determine antibacterial action of GA and VAN in combination against 23 enterococci

Note that: a. FICI value less than or equal to 0.5 indicates synergistic activity, FICI less than or equal to 0.5 indicates additive effect, and FICI > 4 indicates antagonism.

As shown in fig. 2, synergy was also observed in the time sterilization curve experiments of GA and VAN combinations. Vancomycin (4 μg/mL) showed poor bacteriostatic activity when used alone at sub-MIC concentrations, with no improvement in viable count for all test strains at 24 hours compared to the growth control. GA shows weak bacteriostatic action at concentration of 0.5 mug/mL, inoculationBacterial proliferation was evident 8 hours later. When used in combination, a strong synergistic effect was observed on all four test strains, with a significant 2log reduction in viable count compared to VAN alone ₁₀ Colony counts of enterococcus faecalis ATCC 700802, 09-9, enterococcus faecium ATCC 700221 and 12-1 strains were respectively 3.8,3.9,4.2,2.5 log reduced over CFU/mL ₁₀ CFU/mL。

2.3 Synergy of GA and VAN in a mouse multiple organ infection model

Bacterial load in the heart, liver, lung and kidney of mice was determined by colony counting. As shown in fig. 3, GA in combination with VAN significantly reduced bacterial load in mouse organs. Colony counts of heart, liver, lung and kidney were reduced by 1.08, 2.05, 1.20 and 0.77 log, respectively, in the combination treatment group compared to the VAN group ₁₀ CFU/mL。

2.4 Inhibition of topoisomerase IV by GA

Observed under scanning electron microscopy (fig. 4), GA treatment resulted in excessive accumulation of bacterial cell walls around the dividing furrows and formation of a "hamburger" shape, suggesting that bacterial division was impeded.

Based on the morphological changes we observe in bacteria, the important role of type II topoisomerase in bacterial cell division, and the relatively rigid planar structure of GA, we speculate that GA may act on type II topoisomerase in enterococci.

Bacterial gyrase and topoisomerase IV belong to the class II topoisomerase, are critical for bacterial DNA replication and affect their physiological processes including cell division. The main function of DNA gyrase is to introduce negative supercoils before the replication fork, while topoisomerase IV is responsible for the entanglement generated during the de-swirling replication.

The inhibitory activity of GA against commercially available Staphylococcus aureus gyrase and topoisomerase IV is shown in FIG. 5. The topoisomerase II inhibitors ciprofloxacin and neomycin were used as positive controls. Ciprofloxacin inhibits gyrase and topoisomerase IV at 3 μm, while novobiocin inhibits gyrase and topoisomerase IV at 0.01 μm and 1 μm, respectively. GA had no inhibition on DNA gyrase up to 100. Mu.M, but completely inhibited topoisomerase IV at 0.3. Mu.M, reducing the level of supercoiled plasmid pBR 322. GA showed stronger activity against topoisomerase IV than the positive control ciprofloxacin and novobiocin. These results indicate that topoisomerase IV may be one of the targets of GA anti-enterococcus activity.

Topoisomerase IV is ParC ₂ ParE ₂ Is regulated in an ATP-dependent manner. The ParC subunit is the target of quinolones, however bacterial resistance to quinolones and fluoroquinolones is increasing. Novobiocin is the only ParE inhibitor marketed so far that competitively inhibits the atpase activity of ParE, however, toxicity limits its clinical use. The ParE subunit is used as a target point, and the development of the novel antibacterial agent has solid theoretical basis and feasibility. To elucidate whether GA is an inhibitor of ParE, its inhibitory activity on atpase was evaluated. FIG. 6 shows the IC of GA and the positive control novobiocin on ParE ATPase Activity ₅₀ Values and are expressed as the average of three independent measurements. Inhibition of ParE subunit of Staphylococcus aureus by GA (IC ₅₀ =1.68 μg/mL) stronger than neomycin (IC ₅₀ =5.89 μg/mL）。

To further confirm the interaction of GA and enterococcus ParE, we expressed and purified the enterococcus recombinant protein ParE recombinantly. SPR analysis showed that GA can bind to ParE dose-dependently with a Kd value of 7.87. Mu.M (FIG. 7, A), confirming its specific binding capacity. Furthermore, inhibition of ATPase activity of recombinant expressed enterococcus ParE by GA was evaluated, and GA showed a remarkable inhibition of ATPase activity of enterococcus ParE, IC ₅₀ The value was 6.96. Mu.g/mL (FIG. 7, B).

Molecular docking was performed using Discovery Studio 4.5 software to explore the binding pattern between GA and ParE. As shown in FIG. 8, GA was able to fit well into the active pocket of ParE protein, the docking score was 133.1, there could be four hydrogen bonds between ARG79 (2), ARG138, his118 residues and oxygen atoms in different positions, and furthermore, there could be a Pi anion bond between the benzene conjugated systems of GA and GLU 53. It was demonstrated that GA might act directly on the antibacterial target ParE.

The present invention discloses for the first time that GA has good antibacterial activity against enterococci including VRE strains and can restore VAN activity against VRE in vitro and in vivo. GA is likely to be an antibacterial sensitizer for VAN, providing a new therapeutic option for combating VRE.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

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Claims (4)

1. the medicine composition for resisting drug-resistant enterococcus is characterized in that the active ingredients are gambogic acid and vancomycin.

2. The pharmaceutical composition according to claim 1, wherein the mass ratio of gambogic acid to vancomycin is 1:2-1:32.

3. Use of a pharmaceutical composition according to claim 1 or 2 for the preparation of a biologic against drug-resistant enterococci;

the drug-resistant enterococci comprise vancomycin-resistant enterococci and vancomycin-resistant enterococcus faecium.

4. The use according to claim 3, wherein gambogic acid and vancomycin are administered separately, out of order, or together simultaneously.