Screening methods and applications of traditional Chinese medicine inhibitors against carbapenem-resistant Klebsiella pneumoniae

Abstract

This invention relates to a screening method and application of traditional Chinese medicine inhibitors against carbapenem-resistant Klebsiella pneumoniae, belonging to the field of biomedical technology. Based on a high-throughput screening system for carbapenem-resistant Klebsiella pneumoniae inhibitors constructed using a fluorescent probe for carbapenemase activity detection, the invention successfully screened out mulberry bark as a carbapenemase inhibitor, sanghorn ketone G. When used in combination with meropenem, this inhibitor significantly inhibits the metabolism of meropenem by carbapenem-resistant Klebsiella pneumoniae, greatly enhancing the inhibitory efficiency of meropenem against the strain, providing a highly effective and valuable combination therapy for clinical treatment of carbapenem-resistant Klebsiella pneumoniae infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the screening method and application of traditional Chinese medicine inhibitors against carbapenem-resistant Klebsiella pneumoniae. Background Technology

[0002] Carbapenemases are a class of enzymes that can hydrolyze carbapenem antibiotics. β - Lactamases, which include three classes of enzymes classified by Ambler molecular structure: A, B, and D. Classes A and D are serine enzymes, while class B enzymes are metalloenzymes. They are mainly found in Gram-negative bacteria and are capable of hydrolyzing... β - Lactam drugs: The main mechanism by which bacteria develop carbapenemase is resistance to carbapenem antibiotics.

[0003] Class A carbapenemases have serine residues as their active site, including KPC, IMI, and SME. Class A carbapenemases possess the ability to target all... β It exhibits hydrolytic activity against β-lactam antibiotics, but low hydrolytic activity against cephalosporin antibiotics. Its activity can be inhibited by clavulanic acid and tazobactam. EDTA does not affect the activity of class A carbapenemases.

[0004] Type B carbapenemases are also known as metalloenzymes. β β-lactamases require zinc ions or other heavy metal ions as a catalytic medium for their active site. Type B carbapenemases have broad hydrolytic activity, capable of hydrolyzing penicillins, cephalosporins, and carbapenems, but are less effective against monocyclic lactams. β - Lactam antibiotics have low hydrolytic activity [International Journal of Molecular Sciences, 2015, 16(5): 9654–9692]. Because their active sites depend on metal ions, class B carbapenemases are easily inhibited by EDTA metal chelators. β - Lactamases include the NDM family, IMP family, VIM family, etc. There are no clinically approved inhibitors for metallocarbapenemases [ClinicalMicrobiology Reviews, 2020, 33(2)], which greatly increases the difficulty of treating metallocarbapenemase infections.

[0005] Category D β- Lactamases, also known as oxacillinases (OXA), exhibit potent hydrolytic activity against oxacillin. This enzyme is an extremely large and complex family, with over 500 OXA enzyme subtypes identified. Six of these subtypes possess carbapenemase activity: OXA-23, OXA-24 / 40, OXA-51-like, OXA-58, OXA-143-like, and OXA-235-like. The activities of different OXA subtypes vary considerably, and different studies have described the kinetic properties of the same OXA subtype in very different ways [Clinical Microbiology Reviews, 2014, 27(2): 241.].

[0006] In addition, some ESBL enzymes can be transformed into carbapenemases by changing their active sites. For example, GES enzymes are often described as ESBL enzymes because a single point mutation in the gene gives them the activity of hydrolyzing carbapenem antibiotics [Antimicrobial Agents and Chemotherapy, 2001, 45(9): 2598–2603.]. Most carbapenemases share a common characteristic: they are transferable, meaning they are associated with multiple mobile genetic elements in the genetic environment. This also gives most carbapenemases the ability to be horizontally transferred, enabling them to be transferred between different clones and different species of bacteria.

[0007] Therefore, to better study bacterial resistance to carbapenem antibiotics, and considering the important role of carbapenemases in this resistance, as well as their catalytic characteristics, we need to develop fluorescent probes that can sensitively detect carbapenemase activity. This would allow for rapid identification of carbapenemase activity and drug sensitivity in clinically isolated strains. Furthermore, based on these fluorescent probes, we can construct a high-throughput, highly efficient carbapenemase inhibitor screening platform to discover inhibitors derived from traditional Chinese medicine, thereby delaying the hydrolysis of carbapenem antibiotics, enhancing their antibacterial effects, and curbing carbapenem resistance. Summary of the Invention

[0008] To address the problems existing in the prior art, the present invention aims to provide a method and application for screening traditional Chinese medicine inhibitors against carbapenem-resistant Klebsiella pneumoniae. Based on a fluorescent probe for detecting carbapenemase activity, the invention provides high-throughput screening of highly efficient carbapenemase inhibitors, thereby delaying the hydrolysis of carbapenem antibiotics, enhancing their antibacterial effect, and curbing the development of carbapenem antibiotic resistance.

[0009] The technical solution adopted in this invention is: a method for screening traditional Chinese medicine inhibitors of carbapenem-resistant Klebsiella pneumoniae, comprising the following steps:

[0010] S1. Different traditional Chinese medicine extracts were added to the carbapenem-resistant Klebsiella pneumoniae lysate incubation system.

[0011] S2, then the probe substrate is added to initiate the reaction, and incubated at 37 °C;

[0012] The structural formula of the probe substrate is as follows:

[0013] ;

[0014] S3. Subsequently, solvent was added and the reaction was terminated by vortexing. The supernatant was then taken for fluorescence detection to quantitatively determine the inhibition of different inhibitors on carbapenem-resistant Klebsiella pneumoniae.

[0015] Furthermore, in step S2, the concentration of the probe substrate is 1 / 10 to 10. K m .

[0016] Furthermore, in step S3, the reaction temperature is 20°C. o C to 60 o The incubation system should be between 4.0 and 9.0 pH; the reaction time should be between 5 and 120 minutes.

[0017] Furthermore, the fluorescence signals of the probe substrate and its hydrolysis products were obtained using an excitation wavelength of 660-690 nm and a maximum emission wavelength of 700-760 nm.

[0018] The traditional Chinese medicine inhibitor of carbapenem-resistant Klebsiella pneumoniae was obtained by screening using the above-mentioned screening method. The obtained inhibitor was Sanghuang ketone G.

[0019] Application of traditional Chinese medicine inhibitors against carbapenem-resistant Klebsiella pneumoniae, and the application of lindane G combined with meropenem in the preparation of drugs against carbapenem-resistant Klebsiella pneumoniae.

[0020] The beneficial effects of this invention are as follows: The carbapenem-resistant Klebsiella pneumoniae inhibitor screening system constructed in this invention achieves precise quantification of inhibitory effects using fluorescence detection at specific excitation and emission wavelengths, successfully screening out Sanghuang ketone G, an effective traditional Chinese medicine inhibitor. When this inhibitor is used in combination with meropenem, it can significantly inhibit the metabolism of meropenem by carbapenem-resistant Klebsiella pneumoniae, greatly improving the inhibitory efficiency of meropenem against the strain, and providing a highly efficient and valuable combination therapy for clinical treatment of carbapenem-resistant Klebsiella pneumoniae infection.

[0021] This specific probe substrate can be used to determine the enzymatic activity of carbapenemases in biological systems. The procedure for determining carbapenemase activity is as follows: The hydrolysis reaction of hemicyanine compounds is selected as the probe reaction, and the activity of carbapenemases in various biological samples is determined by quantitatively detecting the amount of metabolites generated per unit time. This method can rapidly identify the carbapenemase activity and drug sensitivity of clinical isolates; it enables high-throughput screening of highly effective carbapenemase inhibitors, delaying the hydrolysis of carbapenem antibiotics, enhancing their antibacterial effects, and curbing carbapenem antibiotic resistance.

[0022] This type of probe exhibits excellent selectivity for carbapenemases, while other hydrolases hardly catalyze the probe reaction. The fluorescence response of carbapenemases is 24 times that of lactamases. Therefore, this probe reaction can selectively detect the enzymatic activity of carbapenemases. This probe is a selective probe for detecting carbapenemase activity, thereby enabling efficient discovery of microorganisms with high carbapenemase expression and in-depth research on carbapenemase-related diseases. An efficient screening system for carbapenemase inhibitors was constructed, and it was found that mulberry bark-derived mulberry senna G exhibits the strongest inhibitory activity against carbapenemases. Combined use with carbapenem antibiotics significantly enhances the antibacterial effect of antibiotics and alleviates drug resistance in resistant strains. Attached Figure Description

[0023] Figure 1 It is the structural formula of the probe substrate.

[0024] Figure 2 It is ACLE-S-HC 1 H-NMR spectrum.

[0025] Figure 3 It is ACLE-S-HC 13 C-NMR spectrum.

[0026] Figure 4 This is a high-resolution mass spectrum of ACLE-S-HC.

[0027] Figure 5 These are the results of the ACLE-S-HC hydrolase screening experiment.

[0028] Figure 6 This is the result of inhibition by multiple inhibitors in the lysate of carbapenem-resistant Klebsiella pneumoniae.

[0029] Figure 7 This is a standard curve diagram of carbapenemase protein concentration determination.

[0030] Figure 8 This is a fluorescence imaging image of carbapenem-resistant Klebsiella pneumoniae inhibited by avibactam sodium. In the image, A shows the fluorescence imaging result of a single colony of carbapenem-resistant Klebsiella pneumoniae, and B shows the quantitative fluorescence result.

[0031] Figure 9 This is a high-throughput screening result of various Chinese herbal extracts regulating carbapenemase activity.

[0032] Figure 10 This is a graph showing the inhibition curve of carbapenemase by lindone G.

[0033] Figure 11 This is a fluorescence imaging image of carbapenem-resistant Klebsiella pneumoniae inhibited by linalool G. In the image, A shows the fluorescence imaging result of a single colony of carbapenem-resistant Klebsiella pneumoniae, and B shows the quantitative fluorescence result.

[0034] Figure 12 This is a result of linalool G inhibiting meropenem metabolism.

[0035] Figure 13 This is a result of linalool G enhancing the antibacterial effect of meropenem. Detailed Implementation

[0036] The following embodiments will further illustrate the present invention, but are not intended to limit the invention.

[0037] This invention provides a selective fluorescent probe for detecting carbapenemases. The probe, as a substrate, can be specifically catalyzed by carbapenemases to generate the corresponding product. The probe substrate structure is as follows:

[0038]

[0039] The probe substrate was named ACLE-S-HC.

[0040] The probe substrate has the characteristics of high selectivity of metabolic enzymes (mainly carbapenemase), easy detection of metabolites, and high sensitivity.

[0041] This invention also provides a selective fluorescent probe for detecting carbapenemase. Using the aforementioned compound as a specific substrate for carbapenemase, a hydrolysis reaction is performed. The activity of carbapenemase in different biological systems (including recombinant carbapenemase expression, microbial preparation solutions, and various microbial cells) is quantitatively determined by measuring the substrate elimination rate or the product formation rate per unit time. The specific determination method is as follows:

[0042] The concentration of the probe substrate in the system is selected to be 1 / 10~10. K m ; Preferred substrate concentration for single-point determination K m .

[0043] In buffer solutions such as PBS, the reaction temperature is 20°C. o C to 60 o Between C, 37 is preferred. oC represents the optimal reaction time; the pH of the incubation system should be between 4.0 and 9.0, with pH 7.4 being the preferred optimal reaction pH.

[0044] The reaction time is 5 to 120 minutes. The reaction is terminated when the corresponding hydroxylated products of the above substrates reach the limit of quantitation and the substrate conversion rate does not exceed 20%.

[0045] The amount of substrate reduction or product generation per unit time is used as an evaluation index for carbapenemase activity.

[0046] The present invention provides a method and application for screening traditional Chinese medicine inhibitors of carbapenem-resistant Klebsiella pneumoniae. The probe substrate has no fluorescence, but its hydroxylation product has strong fluorescence properties. A fluorescence detector can be used to achieve rapid and sensitive detection of both the substrate and the product. The fluorescence detection conditions for the hydrolysis product are as follows: excitation wavelength 660-690 nm and maximum emission wavelength 700-760 nm.

[0047] This specific probe substrate is a fluorescent probe, which is not easily affected by the biological matrix and impurities in the carbapenemase activity detection process. It can be used for the quantitative determination of the activities of various recombinant carbapenemases, microbial preparation solutions, and carbapenemases and their isoenzymes in various microbial cells. It can also be used as a probe substrate for whole microbial carbapenemases to assess individual and species differences in carbapenemases. The fluorescent detection method of this probe substrate and its hydrolysis metabolites can also be used for the rapid screening of reversible and irreversible inhibitors, activators, and inducers of carbapenemases, and for the quantitative evaluation of their activity regulation and inducible expression capabilities.

[0048] The study investigated the effects of carbapenem-resistant Klebsiella pneumoniae incubation. Through hydrolytic enzyme selectivity analysis, specific inhibition experiments, and enzyme reaction kinetics, it was demonstrated that hemicyanine compounds can be specifically metabolized by carbapenemases to generate hydrolysis products. Further investigation using molecular docking and other chemical calculation methods, visualization analysis of endogenous microbial carbapenemases, and high-throughput screening of inhibitors revealed that this metabolic reaction exhibits very high specificity.

[0049] As a selective fluorescent probe substrate for highly specific carbapenemases, this compound can be used to detect carbapenemase activity, and is particularly suitable for determining the activity of carbapenemases produced by bacterial, insect cell, clinical isolates and yeast clonal expression systems, as well as for calibrating the activity of carbapenemases in microsomes, S9 and other preparations from various microbial sources.

[0050] Example 1. Synthesis of compound ACLE-S-HC.

[0051] (1) Synthesis method of ACLE-2:

[0052] Pyridine (0.6 mL, 7 mmol) and GCLE (2.4 g, 5 mmol) were added to an anhydrous CH2Cl2 suspension containing PCl5 (1.5 g, 7 mmol) at 0 °C, and the mixture was stirred at 0 °C for 2 hours. After cooling to -40 °C, MeOH was slowly added dropwise, and the mixture was stirred for another 0.5 hours. After removing the solvent using a rotary evaporator, water (3 mL), ethyl acetate (12 mL), and diethyl ether (100 mL) were added to the residue sequentially with vigorous stirring. The precipitate was collected by filtration, washed with diethyl ether, and dried under vacuum to obtain crude ACLE, which could be used for the next step without further purification. NaNO2 (0.5 g, 7 mmol) and water (30 mL) solution were added to a CH2Cl2 solution (30 mL) containing ACLE. After cooling to 0 °C, 1 MH2SO4 (5.2 mL) was added dropwise with vigorous stirring, and the resulting reaction solution was continuously stirred at 0 °C for 1 hour. Separate the organic layer and extract the aqueous layer three times with CH2Cl2. Wash the combined organic layers with brine, dry with MgSO4, and filter to obtain a yellow solution of ACLE-1 (shaking carefully).

[0053] Methanol (12 mL) and 1.3 g of TsOH acid were added to the solution at 0 °C, and the mixture was brought back to room temperature and stirred at room temperature for 3 h. After the reaction was complete, the reaction mixture was concentrated and diluted with ethyl acetate. The organic layer was washed with water and brine, dried over MgSO4, and then evaporated to dryness. Finally, the mixture was purified by silica gel column chromatography to give compound ACLE-2 as a white solid colorless oil (0.33 g, total yield of 17% in three steps). 1 H NMR (600 MHz, Chloroform- d ) δ 7.38 (d, J = 8.6 Hz, 2H), 6.90 (d, J = 8.6 Hz, 2H), 5.30 (d, J = 11.8 Hz, 1H), 5.22 (d, J = 11.8 Hz, 1H), 4.70 (d, J = 1.8 Hz, 1H), 4.52 (d, J = 1.8 Hz, 1H), 4.42 (d, J = 11.8Hz, 1H), 4.31 (d, J = 11.9 Hz, 1H), 3.81 (s, 3H), 3.65 (d, J= 18.1 Hz, 1H), 3.54 (s, 3H), 3.40 (d, J = 18.1 Hz, 1H).

[0054]

[0055] (2) Synthesis method of ACLE-S-OH:

[0056] K₂CO₃ (42 mg, 0.3 mmol) and ACLE-2 (115 mg, 0.3 mmol) were dissolved in anhydrous acetonitrile. The mixture was stirred at room temperature for 0.5 h, and 4-mercaptobenzyl alcohol (50 mg, 0.36 mmol) was added, followed by stirring at room temperature for 3 h. After the reaction was complete, the reaction mixture was concentrated and purified by silica gel column chromatography to give compound ACLE-S-OH as a grayish-white solid (125 mg, 0.255 mmol, yield 85%). 1 H NMR (600 MHz, Chloroform- d ) δ 7.33(d, J = 8.6 Hz, 2H), 7.28 (d, J = 8.3 Hz, 2H), 7.19 (d, J = 8.3 Hz, 2H), 6.88(d, J = 8.7 Hz, 2H), 5.12 (q, J = 11.9 Hz, 2H), 4.59 (s, 3H), 4.48 (d, J =1.7 Hz, 1H), 4.10 (d, J = 13.6 Hz, 1H), 3.81 (s, 3H), 3.77 (d, J = 13.6 Hz, 1H), 3.62 (d, J = 17.6 Hz, 1H), 3.51 (s, 3H), 3.35 (d, J = 17.7 Hz, 1H). 13 CNMR (150 MHz, Chloroform- d) δ 161.41, 161.12, 159.86, 140.58, 132.20, 130.59,127.55, 127.07, 126.41, 125.74, 113.96, 90.15, 67.76, 64.73, 58.25, 56.65,55.33, 37.37, 29.57.

[0057]

[0058] (3) Synthesis method of ACLE-S-HC:

[0059] Iodine (76 mg, 0.3 mmol) was added to an anhydrous dichloromethane solution of triphenylphosphine (78 mg, 0.3 mmol). The mixture was stirred at room temperature for 0.5 h, then imidazole (20 mg, 0.3 mmol) was added, followed by stirring at room temperature for 15 min. ACLE-S-OH (97 mg, 0.2 mmol) was added, and the mixture was stirred for another 30 min. After the reaction was complete, saturated sodium thiosulfate was added to terminate the reaction. The organic layer was separated, washed with brine, and dried over MgSO4. The organic layer was concentrated and purified by chromatographic analysis on a flash column to give the desired compound ACLE-SI (100 mg, 0.174 mmol, yield = 87%) as a yellow oil. A mixture of the above product K2CO3 (42 mg, 0.3 mmol) and HC (78 mg, 0.15 mmol) in anhydrous acetonitrile was stirred at room temperature for 16 h. The reaction mixture was concentrated and purified by flash column chromatography to give the desired compound ACLE-S-HC-PMB (52 mg, 0.059 mmol, yield = 35%) as a blue solid. ACLE-S-HC-PMB (20 mg, 0.02 mmol) was dissolved in 1.5 mL of CH₂Cl₂ at 0 °C, and TFA (1.2 mL) and anisole (0.3 mL) were added. The mixture was stirred at 0 °C for 1 h. Purification by RP-HPLC on a C18 column gave the compound ACLE-S-HC (5 mg, yield = 27%) as a blue solid. 1 H NMR (600 MHz, Methanol-) d 4) δ 8.77 (d, J = 14.9 Hz, 1H), 7.68 (d, J = 7.5 Hz, 1H), 7.54 (dt, J= 15.2, 7.9 Hz, 2H), 7.50 – 7.41 (m, 5H), 7.39 (s, 1H), 7.08 (d, J = 2.3 Hz, 1H), 7.03 (dd, J = 8.6, 2.4 Hz, 1H), 6.52 (d, J = 14.9 Hz, 1H),5.26 (s, 2H), 4.70 (d, J = 1.6 Hz, 1H), 4.50 (d, J = 1.6 Hz, 1H), 4.36 – 4.30(m, 3H), 4.25 (d, J = 13.5 Hz, 1H), 3.92 (d, J = 13.6 Hz, 1H), 3.69 (d, J =17.5 Hz, 1H), 3.49 (s, 3H), 3.42 (d, J = 17.4 Hz, 1H), 2.81 – 2.76 (m, 2H),2.76 – 2.70 (m, 2H), 1.99 – 1.89 (m, 2H), 1.83 (s, 6H), 1.08 (t, J = 7.4 Hz,3H). 13 C NMR (150 MHz, Methanol- d 4) δ 179.42, 163.67, 163.41, 163.19, 155.86,147.24, 143.58, 143.13, 137.31, 136.00, 135.23, 135.19, 133.38, 130.28,130.21, 129.36, 128.77, 128.52, 128.49, 123.90, 117.49, 115.73, 115.32,114.01, 104.86, 102.91, 91.33, 71.37, 58.41, 58.03, 52.10, 47.58, 37.79,32.81, 30.12, 30.05, 28.44, 25.10, 22.31, 21.71, 11.63. HRMS (ESI, m / z): [M] + Theoretical value for C 43 H 43N2O6S2 + , 747.2557, measured value 747.2566. [M-H+Na] + C 43 H 42 N2NaO6S2 + The theoretical value is 769.2377, and the measured value is 769.2381.

[0060]

[0061] Compound ACLE-S-HC 1 H-NMR spectrum, 13 C-NMR spectra and high-resolution mass spectra, such as Figure 2 , Figure 3 , Figure 4 As shown.

[0062] Example 2. In vitro determination of the catalytic selectivity of carbapenemase.

[0063] Incubate the probe ACLE-S-HC (10 μM) with the following enzyme: β -Lactamase ( β -lactamase (Blac), elastase, carboxylesterase 1c (CE1c), and carboxylesterase 2 (CE2). β -glucosidase ( β -Glucosidase, β -Glc), glucuronidase ( β -Glucuronidase, β -Glu), carbonic anhydrase (CA), catalase, dipeptidyl peptidase-4 (DPP4), carbapenemase, including CE1c and CE2. β - The concentrations of Glc and Catalase were 10 μg / mL; Elastase, β The concentrations of Glu, CA, and DPP4 were 1 U / mL; the concentration of Balc was 0.01 U / mL; and the concentration of carbapenemase was 20 ng / μL. The reaction was incubated at 37 °C for 60 minutes, followed by 100 μL of ice-cold acetonitrile and vortexing to terminate the reaction. 200 μL of the supernatant was then used for fluorescence detection.

[0064] from Figure 5As can be seen, this probe exhibits good selectivity for carbapenemases, while other enzymes hardly catalyze the probe reaction. Therefore, this probe reaction can selectively detect the enzymatic activity of carbapenemases.

[0065] Example 3. Inhibition test of multiple inhibitors in carbapenem-resistant Klebsiella pneumoniae lysate.

[0066] Different inhibitors were added to the incubation system of carbapenem-resistant Klebsiella pneumoniae lysate (20 ng / μL), including loperamide (LPA), a selective inhibitor of carboxylesterase 2; sivelestat sodium, an elastase inhibitor; bis(4-nitrophenyl) phosphate (BNPP), a broad-spectrum carboxylesterase inhibitor; and avibactam sodium, a carbapenemase inhibitor. The four inhibitors were pre-incubated with the enzyme-sourced carbapenem-resistant Klebsiella pneumoniae lysate at 37 °C for 3 minutes, followed by initiation with the probe ACLE-S-HC (final concentration 10 μM), incubation at 37 °C for 60 minutes, and then termination of the reaction by vortexing with 100 μL of ice-cold acetonitrile. 200 μL of the supernatant was collected for fluorescence detection. The control sample without inhibitors was set as 100%, and the percentage residual activity of each inhibitor group was calculated.

[0067] from Figure 6 As can be seen, the carbapenemase inhibitor avibactam sodium exhibited a very significant inhibitory effect on the metabolism of ACLE-S-HC in bacterial lysates, while other enzyme inhibitors hardly interfered with the metabolic reactions of ACLE-S-HC. The experimental results further confirm the enzyme selectivity of ACLE-S-HC.

[0068] Example 4. Determination of standard curve for carbapenemase protein concentration.

[0069] The experiment was performed using a 96-well plate on a microplate reader. ACLE-S-HC (10 μM) was incubated with different concentrations of carbapenemase (0, 5, 8, 10, 12, 15, 18 and 20 ng / mL) at 37 ℃ for 60 min. Then, 100 μL of ice-cold acetonitrile was added, and the reaction was terminated by vortexing. 200 μL of the supernatant was taken for fluorescence detection to determine the changes in fluorescence spectra in the reaction solution with different concentrations of carbapenemase. The linear regression equation between the fluorescence intensity of the product and the protein concentration was calculated. Figure 7 The left side of the image shows the fluorescence intensity curves under different concentrations of carbapenemase catalysis, while the right side shows the linear relationship between carbapenemase concentration and fluorescence intensity. This result indicates that the probe substrate has a broad linear range and can accurately quantify the content of carbapenemase.

[0070] Example 5. Visual analysis of endogenous carbapenemases in carbapenem-resistant Klebsiella pneumoniae.

[0071] Carbapenem-resistant Klebsiella pneumoniae was inoculated onto solid LB agar plates and incubated at 37°C. o Incubate at C20 until single colonies appear. Then, evenly spray the colonies with an inhibitor (final concentration of 10 μM) and incubate at 37°C. o After incubating in a C24 incubator for 0.5 hours, a buffer solution containing ACLE-S-HC (final concentration 10 μM) was evenly sprayed onto the colonies. Then, the colonies were incubated at 37°C. o Incubate at 37°C for 0.5 hours, then repeat the spraying once more, and then continue incubation at 37°C. o Incubate at C for 0.5 hours. After completing the above steps, take a picture using a fluorescence imager. Figure 8 and Figure 11 In the figure, A represents the fluorescence imaging result of a single colony of carbapenem-resistant Klebsiella pneumoniae, indicating that the probe can be used for the visualization analysis of microbial endogenous carbapenemase activity and can accurately quantify the content of carbapenemase. Figure 8 and Figure 11 B represents the quantitative fluorescence result. After treatment with avibactam sodium or morinda G, the fluorescence intensity decreased significantly and showed a highly statistically significant difference compared to the probe group (****). P <0.0001), avibactam sodium or morinda G can effectively suppress the fluorescence signal of this probe.

[0072] Example 6. High-throughput screening of carbapenemase regulators from traditional Chinese medicine.

[0073] Screening system: pH 7.4 buffer (100 mM), ACLE-S-HC final concentration 10 μM, traditional Chinese medicine extract final concentration 100 μg / mL, carbapenemase final concentration 20 ng / μL. Assay and control groups were set up separately. After mixing the traditional Chinese medicine extract, carbapenemase, and buffer, the mixture was incubated at 37°C. o Pre-incubate at C for 3 minutes with shaking, then add probe ACLE-S-HC to the sample tube to initiate the reaction. After 60 minutes, add 100 µL of ice-cold acetonitrile to the reaction system, shake vigorously, and terminate the reaction. Centrifuge at 4°C using a high-speed refrigerated centrifuge. o C, 20000 × g Under the specified conditions, after high-speed centrifugation for 10 minutes, the supernatant was collected for fluorescence analysis. The percentage of residual activity of the analyte group was calculated to evaluate the inhibitory ability of the analyte on carbapenemase. Figure 9 This is a high-throughput screening result of traditional Chinese medicine ethanol extracts regulating carbapenemase activity.

[0074] Example 7. Inhibitory activity curve of linalool G against carbapenemase.

[0075] Screening system: 100 mM buffer at pH 7.4, ACLE-S-HC final concentration 10 μM, carbapenemase final concentration 20 ng / μL, and different concentrations of linalool G at 37°C. o After incubation at C for 60 minutes, add 100 µL of ice-cold acetonitrile to the reaction system, shake vigorously, and then terminate the reaction; centrifuge at 4°C using a high-speed refrigerated centrifuge. o C, 20000 × g Under the specified conditions, after high-speed centrifugation for 10 minutes, the supernatant was collected for fluorescence analysis. The percentage residual activity of linalool G at each concentration was calculated, and the inhibition curve of linalool G on carbapenemase was fitted. Figure 10 The results show the inhibition curve of linalool G on carbapenemase, with its half-maximal inhibitory concentration (IC50) being... 50 The value was 1.322 ± 0.068 μmol / L.

[0076] Example 8. Inhibitory effect of linalool G on meropenem metabolism.

[0077] Phellinus linteus G at a final concentration of 20 μM was mixed with 60 ng / µL of carbapenem-resistant Klebsiella pneumoniae lysate and 4 µg / mL of meropenem at 37°C. o Incubate at C for the corresponding times (30 minutes, 1 hour, 2 hours, 4 hours, 12 hours), then terminate the reaction with 100 µL of methanol. o C, 20000 × g Under the specified conditions, after high-speed centrifugation for 220 minutes, the supernatant was collected, and the sample was analyzed by UPLC-MS. Figure 12 This study investigated the inhibitory effect of lindane G on meropenem metabolism in carbapenem-resistant Klebsiella pneumoniae. The results showed that meropenem, when used alone, exhibited extremely rapid clearance, reaching 50% at the half-life time point and maintaining 100% clearance after approximately 4 hours. However, when combined with lindane G, the clearance rate of meropenem significantly decreased, with only about 50% clearance at 12 hours. This indicates that lindane G effectively inhibits the metabolism of meropenem by this bacterium.

[0078] Example 9. Inhibitory effect of lindane G combined with meropenem on carbapenem-resistant Klebsiella pneumoniae.

[0079] Prepare carbapenem-resistant Klebsiella pneumoniae in the logarithmic growth phase and inoculate it into sterile 96-well plates, using 100 µL of bacterial solution per well (approximately 1 × 10⁻⁶). 5 Add 1 µL of serially diluted linalool G solution and 1 µL of serially diluted meropenem solution to each well (CFU). The control group received no antibiotics or linalool G. At 37°C... oIncubate in a C incubator for 18 hours, then measure the absorbance at 600 nm using a microplate reader to calculate the inhibitory activity of each well. Figure 13 This study presents the inhibitory results of lindone G combined with meropenem on carbapenem-resistant Klebsiella pneumoniae. When the meropenem concentration was 0 μg / mL, the inhibition rate of lindone G at all concentrations was extremely low (the highest being only 3.87%). As the meropenem concentration increased, the inhibition rate after combining with lindone G gradually increased. When the meropenem concentration reached 4 μg / mL, the inhibition rate of meropenem alone was less than 62%. The inhibition rate gradually increased with the increase of lindone G dosage, and the inhibition rate approached 100% when the lindone G concentration was ≥16 μg / mL. This indicates that lindone G enhanced the inhibitory effect of meropenem on bacteria.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for screening traditional Chinese medicine inhibitors of carbapenem-resistant Klebsiella pneumoniae, characterized in that, Includes the following steps: S1. Different traditional Chinese medicine extracts were added to the carbapenem-resistant Klebsiella pneumoniae lysate incubation system. S2, then the probe substrate is added to initiate the reaction, and incubated at 37 °C; The structural formula of the probe substrate is as follows: ； S3. Subsequently, solvent was added and the reaction was terminated by vortexing. The supernatant was then taken for fluorescence detection to quantitatively determine the inhibition of carbapenemase in carbapenem-resistant Klebsiella pneumoniae by different Chinese herbal extracts.

2. The screening method according to claim 1, characterized in that: In step S2, the concentration of the probe substrate is 1 / 10 ~ 10. K m .

3. The screening method according to claim 1, characterized in that: The fluorescence signals of the probe substrate and its hydrolysis products were obtained by using an excitation wavelength of 660-690 nm and a maximum emission wavelength of 700-760 nm.

4. Application of Sanghuang ketone G in the preparation of carbapenemase inhibitors in carbapenem-resistant Klebsiella pneumoniae.

5. Application of the combination of lindane G and meropenem in the preparation of inhibitors of carbapenem-resistant Klebsiella pneumoniae.

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