Screening method and application of campylobacter function-enhanced multi-drug-resistant efflux pump variant RE-CmeABC inhibitor

Abstract

The invention belongs to the technical field of antibacterial efflux pump inhibitors, and particularly relates to a screening method and application of a campylobacter function enhanced multi-drug-resistant efflux pump variant RE-CmeABC inhibitor. The method comprises the following steps: firstly, predicting and constructing a key efflux protein RE-CmeB homologous model in RE-CmeABC based on an RE-CmeB protein sequence in an NCBI (National Center of Biotechnology Information) database to obtain an RE-CmeB screening model, then docking a plurality of antibacterial drugs capable of being efflux by the RE-CmeABC with the obtained screening model, and finally screening a molecular docking result. The invention discloses a method for preparing a multi-drug-resistant efflux pump variant RE-CmeABC inhibitor, which is characterized in that the multi-drug-resistant efflux pump variant RE-CmeABC inhibitor ZINC338037, ZINC103293168 and ZINC5999026 with enhanced functions are found, and further drug verification tests are carried out on the multi-drug-resistant efflux pump variant RE-CmeABC inhibitor A systematic efflux pump inhibitor screening scheme is summarized, and potential lead compounds for campylobacter infection prevention and treatment are found, so that a foundation is laid for subsequent research and development of efflux pump inhibitors, and the method has important significance in solving the global drug-resistant bacterium problem.

Description

Technical Field

[0001] The present invention belongs to the technical field of antimicrobial efflux pump inhibitors, and in particular relates to a screening method and application of a Campylobacter function-enhanced multidrug-resistant efflux pump variant RE-CmeABC inhibitor. Background Art

[0002] Campylobacter coli is an important foodborne and zoonotic pathogen. In recent years, Campylobacter (Campylobacter in this application refers to Campylobacter coli) has become increasingly resistant to multiple antimicrobial drugs, which not only endangers the healthy development of the aquaculture industry, but also threatens human health and public safety. Efflux pump-mediated drug efflux is one of the important mechanisms of bacterial resistance. The efflux pump CmeABC is mainly composed of CmeA (periplasmic membrane fusion protein), CmeB (inner membrane transport protein), and CmeC (outer membrane channel protein). It is an inherent resistance mechanism of Campylobacter, which can promote the emergence of fluoroquinolone resistance mutations, and can also synergize with other resistance mechanisms to cause Campylobacter resistance (Antimicrob Agents Chemother. 2003, 47 (1): 390-394; J Antimicrob Chemother. 2006, 58 (6): 1154-1159).

[0003] In previous studies, we discovered a strong variant of the efflux pump CmeABC and named it RE-CmeABC. The amino acid homology of this variant with CmeA and CmeC in CmeABC is greater than 98%, while the amino acid homology of CmeB is only 81%. This variant is stronger than the wild-type CmeABC in terms of efflux of multiple drugs, resulting in enhanced resistance of Campylobacter to multiple antimicrobial drugs. In addition, the prevalence of this variant in Campylobacter is increasing (Engineering, 2020, 6, 34-39; J Antimicrob Chemother. 2021, 76(3): 596-600).

[0004] At present, it is known that there are many compounds that can inhibit the efflux function of efflux pumps, such as phenylalanine-arginine β-naphthylamide (PaβN), compound SILA421, verapamil, milbemycin, reserpine, etc. Among them, PaβN is the earliest discovered broad-spectrum inhibitor, which not only inhibits the three main RND efflux pumps (MexAB-OprM, MexCD-OprJ and MexEF-OprN) in Pseudomonas aeruginosa, but also inhibits the AcrAB-TolC efflux pump of Escherichia coli. Compound SILA421 is a compound that has a killing effect on multidrug-resistant and extensively drug-resistant bacteria. It not only inhibits sensitive tuberculosis bacteria, but also inhibits the efflux pump activity of tumor cells, and does not show toxicity to human cells, which is very promising (Int J Antimicrob Agents, 2009, 33(5): 479-82). However, with the deepening of research, the defects of related efflux pump inhibition have gradually been exposed. For example, the two stubborn cationic groups of PAβN are still highly toxic after modification and other optimizations; SILA421 has maintained good effects in terms of toxicity, but has stopped at in vivo trials, etc. Although most of them have broad-spectrum properties, their own structure is a defect that must be faced in clinical practice.

[0005] Traditional Chinese medicine has a long history in my country, and most of them are derived from plants. They are less toxic and easier to obtain, and are a natural inhibitor material library. Literature reports that there are currently efflux pump inhibitors for Pseudomonas aeruginosa, such as coumaric acid and its derivatives (Comb Chem High Throughput Screen, 2016, 19(9): 705-713). In addition, a small number of natural products such as rue (Planta Med, 2007, 73(15): 1554-7) and plumbagin (Int J Antimicrob Agents, 2011, 37(2): 156-61) have stood out in preliminary efflux pump inhibitor screening tests.

[0006] There are three common research methods for efflux pump inhibitors. The first is the microbial sensitivity test (AST) of efflux pump inhibitors. Conventional AST using intact cells can directly observe the inhibitory activity of efflux pump inhibitors on efflux pumps. Currently, the development or verification of efflux pump inhibitors using clinical isolates is the most commonly used method for researchers. The second is computer-aided drug design based on SAR, which is to rationally design and reasonably modify the structure of lead compounds based on molecular mechanisms and structure-activity relationships (SAR). The third is virtual screening of efflux pump inhibitors from compound libraries, which is to search for molecular structures with potential activity from natural product databases. Virtual screening is currently the most economical and effective method for discovering lead compounds. This ligand-based or high-throughput virtual screening technology has been continuously used to screen efflux pump inhibitors against RND from natural compound libraries (PLoS One, 2014, 9(7): e101840; Comb Chem High Throughput Screen, 2016, 19(9): 705-7713).

[0007] Through a large amount of literature analysis, the present invention established a RE-CmeB protein model, used virtual screening techniques such as molecular docking, and discovered the Campylobacter function-enhanced multidrug-resistant efflux pump variant RE-CmeABC inhibitor in the natural product database, and evaluated its biological activity and safety in vitro, providing broad prospects for the research and development of clinical antibacterial drugs for drug-resistant Campylobacter. Summary of the invention

[0008] The purpose of the present invention is to provide a screening method and application of a Campylobacter function-enhanced multidrug-resistant efflux pump variant RE-CmeABC inhibitor, so as to provide a solution for controlling bacterial resistance and thus ensure public health safety.

[0009] The design ideas of the present invention are as follows: first, based on the protein sequence in the NCBI database and the SWISS MODLE website, the homology model of the key efflux protein RE-CmeB in RE-CmeABC is predicted and constructed, and the protein model is modified and optimized using GalaxyRefine and comprehensively evaluated to obtain the RE-CmeB screening model. Through the AutoDock Vina software, a variety of antibacterial drugs that can be effluxed by RE-CmeABC are molecularly docked with the RE-CmeB screening model to determine the specific binding pocket of RE-CmeB.

[0010] Then, a natural product database was created based on ZINC15 data, and small molecule compound screening was performed. The ligands were screened using VINA software, and the semi-flexible docking method was selected. According to the VINA scoring standard Affinity, the part with a stronger binding force of ≤-7kcal / mol was selected. The molecular docking results were screened using ADMET and drug-like analysis, and the affinity was sorted using molecular dynamics simulation to gradually narrow the scope. Finally, compounds that can serve as inhibitors of the functionally enhanced multidrug-resistant efflux pump variant RE-CmeABC were identified and further drug trials were conducted for verification.

[0011] To achieve the above object, the technical solution created by the present invention is implemented as follows:

[0012] The present invention first provides a method for screening inhibitors of Campylobacter function-enhanced multidrug-resistant efflux pump variant RE-CmeABC, comprising the following steps:

[0013] 1) Based on the RE-CmeB protein sequence in the NCBI database and the SWISS MODLE website, the homology model of the key efflux protein RE-CmeB in RE-CmeABC was predicted and constructed, and then modified, optimized and comprehensively evaluated to obtain the RE-CmeB screening model;

[0014] 2) docking the multiple antimicrobial drugs that can be effluxed by RE-CmeABC with the screening model obtained in step 1) to determine the specific binding pocket of RE-CmeB;

[0015] 3) Create a natural product small molecule ligand database based on ZINC15 data, use the screening model binding pocket obtained in step 2) as the receptor, use the software VINA to perform molecular docking between the obtained protein screening model and the small molecules in the natural product database, and then perform virtual screening such as drug-like screening, ADMET screening and molecular dynamics simulation to achieve screening of small molecule compounds, and finally screen out compounds that can serve as inhibitors of the functionally enhanced multidrug resistance efflux pump variant RE-CmeABC, and conduct further drug validation tests on them.

[0016] Specifically, in step 1), the RE-CmeB protein sequence is shown as SEQ ID NO.1.

[0017] Specifically, in step 2), the antibacterial drug is one or more of florfenicol, ciprofloxacin, erythromycin, tetracycline, and gentamicin.

[0018] Specifically, in step 2), the specific binding pocket of RE-CmeB was determined to be 20 key amino acids: SER47, GLN88, LYS90, ARG131, ILE179, SER177, THR129, ASP152, ILE137, ASN181, SER279, ILE292, GLU275, GLN276, MET571, VAL606, LEU608, PHE611, LEU613, and PHE626.

[0019] Specifically, the molecular docking method in step 3) is: first calculate the energy according to the structure and force field parameters of the small molecule, then search all possible binding sites around the active pocket of the screening model, and select a semi-flexible docking method; according to the scoring standard Affinity of the software VINA, select -7 kcal / mol as the threshold, exclude small molecule ligands with scores greater than -7 kcal / mol, and select the part with stronger binding force when the score is ≤-7 kcal / mol.

[0020] Specifically, during drug-likeness screening in step 3), drug-likeness analysis based on Lipinski's "Rule of Five" (RO5) was performed on 85,885 compounds obtained through virtual screening, and potential lead compounds obtained through molecular docking were further screened.

[0021] Specifically, in step 3), the ADMET module of the GHDDI-AIDD platform is used to perform ADMET screening on the results of drug-like screening; further preferably, water solubility, blood-brain barrier penetration, CYP2D6 binding, hepatotoxicity, intestinal absorption and plasma protein binding are selected for screening.

[0022] Specifically, in step 3), during the docking process, the dielectric constant of the molecular dynamics simulation system is set to 2, the implicit solvent model selects GBMV, the atomic radius is set to the van der Waals radius, and the molecular dynamics simulation is used for affinity ranking.

[0023] Furthermore, based on a general inventive concept, the present invention also provides a Campylobacter function-enhanced multidrug-resistant efflux pump variant RE-CmeABC inhibitor obtained by screening through the above method; the inhibitor is one or a combination of two or more inhibitors selected from ZINC338037, ZINC103293168, and ZINC5999026.

[0024] Preferably, the inhibitor is a combination of two of ZINC338037, ZINC103293168, and ZINC5999026.

[0025] More preferably, the inhibitor is a combination of ZINC338037, ZINC103293168 and ZINC5999026.

[0026] Specifically, the structural formulas of compounds ZINC338037, ZINC103293168, and ZINC5999026 are:

[0027]

[0028] The inhibitor of the present invention is an efflux pump inhibitor with enhanced sensitivity to antimicrobial drugs. Test results show that:

[0029] The Campylobacter function-enhanced multidrug-resistant efflux pump variant RE-CmeABC inhibitor of the present invention can enhance the antibacterial activity of florfenicol, erythromycin, chloramphenicol and tetracycline against Campylobacter containing the multidrug-resistant efflux pump variant RE-CmeABC. When used alone, the strain 11168-RE-CmeABC has a high sensitivity to the small molecule compound ZINC338037. When used in combination, the FICs of the compound and florfenicol, erythromycin, chloramphenicol and tetracycline are 0.2515, 0.2656, 0.28125 and 0.3125 respectively. The test results show that the best effect is achieved when used in combination with florfenicol, which reduces the drug sensitivity of the strain 11168-RE-CmeABC to florfenicol from 16 μg / mL to 4 μg / mL, significantly increasing the antibacterial activity of florfenicol, followed by erythromycin, chloramphenicol and tetracycline, and the MIC values ​​are all reduced by 4 times.

[0030] The results of the efflux inhibition test showed that the combination of compound ZINC338037 and florfenicol can increase its accumulation in the bacteria, which has the effect of reducing drug efflux. The results of the cytotoxicity test showed that the survival rate of African monkey kidney cells (Vero) was 100% at 512μg / mL of compound ZINC338037, and the survival rate of pig kidney cells (PK-15) reached 100% at 32μg / mL. The results of the red blood cell hemolysis test showed that the hemolysis rate of compound ZINC338037 was reduced to 0 at a concentration of 160μg / mL, indicating that ZINC338037 has high selectivity and high safety.

[0031] Furthermore, based on a general inventive concept, the present invention also provides the use of the multidrug-resistant efflux pump variant RE-CmeABC inhibitor in enhancing the antibacterial activity of multiple antibiotics against Campylobacter containing the multidrug-resistant efflux pump variant RE-CmeABC.

[0032] Specifically, the antibiotic is one or more of florfenicol (FFC), ciprofloxacin (CIP), tetracycline (TET), chloramphenicol (CHL) and erythromycin (ERY).

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. The screening method of the present invention is relatively fast and efficient, and can quickly reduce candidate molecules and determine their number, avoiding waste of experimental materials and time. Since the establishment of the screening rules and models is based on the homology model of the key efflux protein RE-CmeB in RE-CmeABC based on the known protein sequence, the final screening results are also verified by experimental data, and the theory and experimental results complement each other, so the screening method and results of the present invention are highly reliable.

[0035] 2. The molecular docking used in the method of the present invention is based on the RE-CmeABC inhibitor, a multidrug-resistant efflux pump variant with enhanced function of Campylobacter, and is highly targeted.

[0036] 3. The antibacterial activity test results of the present invention show that compound ZINC338037 can enhance the antibacterial activity of florfenicol, erythromycin, chloramphenicol, and tetracycline against Campylobacter containing multidrug-resistant efflux pump variant RE-CmeABC, and reduce the MIC of the above antibacterial drugs by 4 times. The results of the efflux inhibition test show that the combination of compound ZINC338037 and florfenicol can increase its accumulation in the bacteria, and has the effect of reducing drug efflux. The results of the toxicity test show that compound ZINC338037 has almost no cytotoxicity and hemolytic toxicity.

[0037] 4. The method of the present invention can discover new efflux pump inhibitors from natural products, and has the potential and value for in-depth research. The present invention summarizes a set of systematic efflux pump inhibitor screening schemes, laying a foundation for the subsequent research and development of efflux pump inhibitors, and is of great significance for solving the current global problem of drug-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The protein model and model screening results; A is the protein model predicted by SWISS MODLE; B is the RE-CmeB protein screening model; C is the binding pocket of the RE-CmeB protein model and antibiotic docking; D is the model scoring results; E is the protein model Ramachandran plot; F is the Z-Score score;

[0039] Figure 2 is the virtual screening result;

[0040] Figure 3The structure diagram of the small molecule compounds screened; A is ZINC338037; B is ZINC103293168; C is ZINC5999026;

[0041] Figure 4 The results of the drug sensitivity test of compound ZINC338037 combined with antibiotics;

[0042] Figure 5 are the results of the efflux inhibition test; A is the accumulation of florfenicol in NCTC 11168 when the compound is used in combination; B is the accumulation of florfenicol in 11168-RE-CmeABC when the compound is used in combination;

[0043] Figure 6 The results of cytotoxicity test; A is African monkey kidney epithelial cell Vero; B is pig kidney cell PK-15;

[0044] Figure 7 This is a red blood cell hemolytic test; Mock is a phosphate buffered saline (PBS) treatment group; Triton X-100 is a 0.5% Triton X-100 detergent treatment group. DETAILED DESCRIPTION

[0045] The present invention is described in further detail below in conjunction with examples, but embodiments of the present invention are not limited thereto. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise defined, all professional and scientific terms used herein are identical in meaning to those skilled in the art.

[0046] Test strains and main reagents

[0047] Test strains: strain NCTC 11168 was purchased from China Industrial Microbiological Culture Collection (CICC); strain 11168-RE-CmeABC (11168-V1) reference (Hong Yao#, Zhangqi Shen#, Yang Wang, FengruDeng, Dejun Liu, Gaowa Naren, Lei Dai, Chih-Chia Su, Bing Wang, Shaolin Wang, Congming Wu, Edward W.Yu, Qijing Zhang*, Jianzhong Shen*, Emergence of a potent multidrug efflux pump variant that enhances Campylobacter resistance to multiple antibiotics, mBio, 2016, 7(5). pii:

[0048] e01543-16.)

[0049] Experimental reagents and culture medium: MUELLER-HINTON AGAR (MHA) and MUELLER-HINTON BROTH (MHB) were purchased from Oxoid; sterile defibrinated sheep blood was purchased from Zhengzhou Yikan Biological; microaerobic gas generation bag was purchased from Mitsubishi of Japan; florfenicol (standard product) was purchased from TMstandard; phosphate buffer was purchased from Sewell Biological.

[0050] Example 1 Establishment of protein model

[0051] Each precursor of RE-CmeB can generate multiple substrate binding pockets, including proximal and distal multi-drug binding sites, and the entrance of its periplasmic cleft is surrounded by amino acids Y658, L660, R710 and D823, which is very important for the specificity and selectivity of RE-CmeB substrates. Therefore, the present invention starts with the RE-CmeB protein sequence and screens RE-CmeABC inhibitors through molecular docking, virtual screening and other steps.

[0052] First, the RE-CmeB protein sequence was obtained from the NCBI database and input into the SWISS MODEL website, and the protein model was obtained using the website.

[0053] The RE-CmeB protein sequence (AOR83335, 1039aa) was obtained from the NCBI database, as shown in SEQ ID NO. 1. The protein model obtained from the SWISS MODEL website (as shown in Figure 1 A).

[0054] Example 2 Modification and optimization of protein model

[0055] Load the obtained protein model file into the software GalaxyRefine: preprocess the input model, including removing water molecules, repairing incorrect side chain conformations, etc., to ensure the integrity and usability of the input model; after the preprocessed model, start model optimization, select the molecular dynamics algorithm, and reduce the energy of the structure by adjusting the position and conformation of atoms to improve the geometric characteristics of the structure; during the optimization process, GalaxyRefine will perform energy evaluation and structural stability analysis on each conformation, including calculating the total energy of the structure, evaluating the reliability and conformation of the conformation, and analyzing structural features such as hydrogen bonds and disulfide bonds. GalaxyRefine will output the optimized protein structure and the corresponding evaluation results, including information on energy and other evaluation indicators. Finally, select the best results of each evaluation, and use PyMOL to view the obtained three-dimensional protein screening model.

[0056] The specific results are as follows: the obtained protein model was modified and optimized using the software GalaxyRefine, the best results of each evaluation were selected, and the obtained three-dimensional protein screening model (such as Figure 1 B).

[0057] Example 3 Establishment of active pockets and small molecule database

[0058] The protein model was established using SWISS MODLE. With the help of GalaxyRefine, multiple models were compared and optimized, and finally the RE-CmeB protein model for molecular docking screening was obtained.

[0059] According to the drug resistance of RE-CmeABC to multiple drugs, the small molecule three-dimensional structures of the corresponding drugs were obtained in the NCBI database, and multiple antibacterial drugs (florfenicol, ciprofloxacin, erythromycin, tetracycline, gentamicin) that can be excreted by RE-CmeABC were docked with the screening model obtained in Example 2 in AutoDock Vina to obtain the active pocket of the protein model.

[0060] For the selection of ligand sources, we chose the more classic database ZINC15 (https: / / zinc15.docking.org / ) based on the currently popular natural products with many advantages such as rich biological activity, sustainability, and structural diversity. 270,549 small molecule structures in the natural product library were downloaded from the ZINC15 database for screening RE-CmeABC inhibitors. In order to facilitate subsequent docking, the three-dimensional structures of all small molecules were selected from mol2.

[0061] The specific results are: Figure 1 D shows the evaluation results of some protein models, and the highest-scoring part is selected. The PDF function defines geometric properties such as bond length, bond angle, and dihedral angle in the protein structure, which directly reflects the quality of the model. In general, the lower the PDF Total Energy of the model, the better the model is optimized under homology constraints and the higher the model credibility. DOPE score is the basis for measuring model quality. The lower the score, the more reliable the model quality. After analyzing multiple models, the optimized model is more in line with the Z-score range ( Figure 1 F), in addition, according to the Laplace diagram results ( Figure 1 E) It can be seen that the number of amino acids in the allowed region accounts for 99.4%, and the proportion of amino acids in the disallowed region accounts for 0.6%. The disallowed region meets the evaluation requirements of the homologous protein model, that is, the proportion of amino acids is <5%.

[0062] After docking multiple small molecule antibiotics using AutoDock Vina, the red area was predicted to be a potential binding pocket (such as Figure 1 C), the binding site of the RE-CmeB protein model was determined based on the docking results of antibiotics: florfenicol, ciprofloxacin, erythromycin, tetracycline, gentamicin, etc. The key amino acid sites that make up the binding pocket are SER47, GLN88, LYS90, ARG131, ILE179, SER177, THR129, ASP152, ILE137, ASN181, SER279, ILE292, GLU275, GLN276, MET571, VAL606, LEU608, PHE611, LEU613, PHE626.

[0063] Example 4 Virtual screening based on molecular docking

[0064] Virtual screening uses Vina (v.1.1.2) software for molecular docking. Vina (v.1.1.2) software automatically calculates the energy of the small molecule based on its structure and force field parameters, and then rotates and translates the small molecule to search for all possible binding sites around it based on the obtained protein model active pocket. All structure file formats are converted to PDBQT format, and the calculation results of the function score are given by calculating factors such as the van der Waals force, electrostatic interaction and hydrogen bond between the protein and molecule at the site; the docking adopts a semi-flexible docking method, that is, RE-CmeB is used as a rigid body and the small molecule ligand is a flexible body. The rotatable bonds in all ligands are sampled, and the remaining parameters are set to default values.

[0065] The molecular docking and virtual screening results are:

[0066] The obtained protein screening model was docked with small molecules in the natural product database using VINA. The Affinity in VINA was used for scoring. According to the rule of thumb: when Affinity ≤ -7 kcal / mol, the binding force is strong. Sort by Affinity score, select -7 kcal / mol as the threshold, remove small molecule ligands with scores > -7 kcal / mol, and screen 85,885 compounds.

[0067] Example 5 Drug Property Screening and ADMET Screening

[0068] In order to ensure the safety and absorption effect of potential small molecule compounds, drug-likeness analysis based on Lipinski's "Rule of Five" (RO5) was performed on them, and potential lead compounds were further screened through molecular docking.

[0069] In order to predict the drugability of small molecule compounds screened by molecular docking, the ADMET module of the GHDDI-AIDD platform was used for screening, and water solubility, blood-brain barrier penetration, CYP2D6 binding, hepatotoxicity, intestinal absorption and plasma protein binding were selected for screening.

[0070] The drug-like properties and ADMET screening results are:

[0071] like Figure 2 As one of the docking results, the small molecule has hydrogen bonding with amino acids ARG131, SER177, and GLY180. The 85,885 compounds obtained by molecular docking were screened by ADMET, and the compounds that did not meet the requirements were filtered out. Finally, 50 potential lead compounds were obtained, as shown in Table 1.

[0072] Table 1 Binding free energy of some small molecule ligands

[0073]

[0074]

[0075] As shown in Table 1, the binding free energy results of some small molecule ligands are presented. Combined with GROMACS software, the drug-likeness and ADMET screening results are sorted according to the strength of binding free energy, and then analyzed subsequently.

[0076] Example 6 Molecular dynamics simulation

[0077] GROMACS was used to establish the CHARMM force field to study the receptor-ligand complex obtained by virtual screening. The setting was carried out under NPT (normal temperature and pressure) conditions, the Langevin temperature control algorithm was used to keep the temperature stable, the PME (plane wave method) was used to deal with long electrostatic interactions, and potential wells were introduced at specific sites where proteins and small molecules bind to keep the protein-small molecule complex stable; since RE-CmeB is a transmembrane protein, the dielectric constant of the simulation system was set to 2, the implicit solvent model selected GBMV (Generalized Born Model with a simple van der Waals term), the atomic radius was set to van der Waals radius (van der Waals radii), and other conditions were set to default.

[0078] The molecular dynamics simulation results are:

[0079] In molecular dynamics simulation, detecting the binding free energy between molecules is an important application, which can show the binding affinity between ligand and protein.

[0080] According to the analysis of the data in Table 1, the higher the binding energy, the easier it is for them to bind to the RE-CmeB receptor protein. We speculate that they may have better potential biological activity. After comprehensively considering the binding free energy strength and molecular dynamics screening results, ZINC338037 (molecular formula C 16 H 14 O4), ZINC103293168 (molecular formula is C 19 H 24 N2O2), ZINC5999026 (molecular formula is C 16 H 14 O5) Three small molecule compounds were evaluated in the next step. The structures of the three compounds are as follows Figure 3 shown.

[0081] Example 7 Drug sensitivity test

[0082] The strain carrying RE-CmeABC was selected for drug sensitivity test, and the antibiotics florfenicol (FFC), ciprofloxacin (CIP), tetracycline (TET), chloramphenicol (CHL) and erythromycin (ERY) were selected as test drugs. The specific steps are as follows:

[0083] (1) Resuscitation of Campylobacter test strain 11168-RE-CmeABC was cultured on MHA agar plates under microaerobic conditions (5% O2, 10% CO2, 85% N2 by volume) for 24 h, then subcultured on new MHA agar plates and cultured for 18 h.

[0084] (2) Scrape an appropriate amount of bacterial cells and add them to 500 μL MHB broth and mix thoroughly. Take 150 μL of bacterial solution and add it to a 90 mm plate containing 15 mL MHB broth to dilute it 100 times. Use a multichannel pipette to mix it.

[0085] (3) Use a multichannel pipette to draw the bacterial solution from the plate and add it to a 96-well plate, 100 μL per well, for a total of 11 columns. Add MHB blank broth to the 12th column as a negative control.

[0086] (4) Add 100 μL of the compound to the first column of the 96-well plate to a final concentration of 1280 μg / mL. Mix by pipetting and then transfer 100 μL of the mixture to the next column for serial dilution. Dilute to column 10 and column 11 as a positive control.

[0087] (5) The 96-well plate was placed in a 42° C. incubator and cultured under microaerobic conditions (5% O 2 , 10% CO 2 , 85% N 2 by volume) for 24 h.

[0088] (6) Result judgment and interpretation: Before reading and reporting the MIC of the tested strain, check whether the bacterial growth in the positive control well is good, whether the negative control well is contaminated, and whether the MIC value of the quality control drug is within the quality control range. The lowest drug concentration in the clear well observed by the naked eye is the MIC of the tested bacteria.

[0089] The results of drug sensitivity test were:

[0090] Campylobacter strains carrying RE-CmeABC were selected for drug sensitivity testing, and the results are shown in Table 2. As a variant of strain NCTC 11168, strain 11168-RE-CmeABC has an overall improved resistance level. In particular, the MICs of florfenicol and chloramphenicol in strains containing RE-CmeB were increased by 32 and 16 times, respectively, compared with the original strain NCTC 11168, and ciprofloxacin, tetracycline, and erythromycin were increased by 8, 4, and 4 times, respectively. In the drug sensitivity test of 11168-RE-CmeABC, the MICs of ZINC338037, ZINC103293168, and ZINC5999026 were 80μg / mL, 160μg / mL, and 160μg / mL, respectively, so ZINC338037 with a lower MIC was selected for the chessboard method drug sensitivity test.

[0091] Table 2 Results of drug sensitivity test

[0092]

[0093] Example 8 Chessboard Drug Susceptibility Test

[0094] The checkerboard assay for combined antibacterial activity was carried out according to the operating procedures of the Clinical and Laboratory Standards Institute (CLSI) of the United States. The microbroth checkerboard dilution method was used to determine the in vitro antibacterial effect of the combination of the compound and antibiotics against the strains. Among them, the antibiotics included florfenicol (FFC), ciprofloxacin (CIP), tetracycline (TET), chloramphenicol (CHL), and erythromycin (ERY).

[0095] Fractional inhibitory concentration indices (FIC) were used for the combined susceptibility test by the microbroth checkerboard dilution method, and the fractional inhibitory concentration index was used as the basis for judging the test results. An FIC index ≤ 0.5 indicates a synergistic effect, meaning that the combined effect of the two drugs is significantly greater than the sum of their individual effects; 0.5 < FIC ≤ 1 indicates an additive effect, meaning that the activity of the two drugs in combination is equal to the sum of their individual antibacterial activities; 1 < FIC ≤ 2 indicates an irrelevant effect, meaning that the activity of the two drugs in combination is equal to their individual activities; FIC > 2 indicates an antagonistic effect, meaning that the combined effect of the two drugs is lower than the individual antibacterial activity.

[0096] FIC = MIC ab / MIC a +MIC ba / MIC b

[0097] MIC a is the minimum inhibitory concentration (MIC) of compound a alone, MIC ab is the minimum inhibitory concentration (MIC) of the combination of compound a and antibiotic b, MIC b is the minimum inhibitory concentration (MIC) of antibiotic b alone, MIC ba is the minimum inhibitory concentration (MIC) of the combination of antibiotic b and compound a, FIC a is the FIC index of compound a, FIC b is the FIC index of antibiotic b. The specific steps are as follows:

[0098] (1) Resuscitate the Campylobacter test strain 11168-RE-CmeABC on an MHA agar plate and culture it for 24 h under microaerophilic conditions (volume ratio: 5% O2, 10% CO2, 85% N2), then subculture it on a new MHA agar plate and culture for 18 h.

[0099] (2) Scrape an appropriate amount of bacterial cells and add them to 500 μL of MHB broth and mix well; take 150 μL of the bacterial solution and add it to a 90 mm plate containing 15 mL of MHB broth to dilute it 100 times, and use a multi-channel pipette to blow and mix well.

[0100] (3) Use a multichannel pipette to draw the bacterial solution from the plate and add it to a 96-well plate, 100 μL per well, for a total of 9 columns. Add bacterial solution to the 11th column as a positive control and add MHB blank broth to the 12th column as a negative control.

[0101] (4) Add 100 μL of compound ZINC338037 to the first column of the 96-well plate to make the final concentration of 80 μg / mL. After pipetting and mixing, pipette 100 μL of the mixture to the next column and dilute it serially to the eighth column. Set the concentration of florfenicol to 7 gradients and use a 2 mL EP tube for serial dilution to obtain 7 concentrations from large to small.

[0102] (5) Add corresponding concentrations of florfenicol from high to low to each layer of the 96-well plate from top to bottom, for a total of 7 rows and 9 columns; at this time, the 8th row of the 96-well plate is the single-drug positive control of compound ZINC338037, and the 9th column is the single-drug positive control of florfenicol.

[0103] (6) The 96-well plate was placed in a 42° C. incubator and cultured under microaerobic conditions (5% O 2 , 10% CO 2 , 85% N 2 by volume) for 24 h.

[0104] The results of the chessboard drug sensitivity test are:

[0105] The results of the chessboard method drug sensitivity test FIC were used to determine whether ZINC338037 and antibiotics had a synergistic effect. When the FIC index was ≤ 0.5, there was a synergistic effect. Figure 4 As shown, when compound ZINC338037 is used in combination with florfenicol, its FIC value is the lowest and the effect is the best, reducing the MIC of 111168-RE-CmeABC to florfenicol from 16μg / mL to 4μg / mL. In addition, the FIC of compound ZINC338037 and erythromycin, chloramphenicol, and tetracycline are all less than 0.5, reducing their MIC by 4 times. The test results show that compound ZINC338037 has a synergistic effect with antibiotics (florfenicol, tetracycline, chloramphenicol, and erythromycin), among which the best effect is achieved when used in combination with florfenicol.

[0106] Example 9 Efflux Inhibition Test

[0107] In order to test whether the combination with ZINC338037 can reduce the efflux pump of florfenicol, a florfenicol accumulation test was conducted, and the efflux pump inhibitor CCCP was used as a positive reference object. The specific steps are as follows:

[0108] (1) Resuscitation of Campylobacter test strains NCTC 11168 and 11168-RE-CmeABC were cultured on MHA agar plates under microaerobic conditions (5% O2, 10% CO2, 85% N2 by volume) for 24 h, then subcultured on new MHA agar plates and cultured for 18 h.

[0109] (2) Inoculate into MHB broth and culture at 42°C, 155 rpm, under microaerobic conditions for 20 h.

[0110] (3) Centrifuge at 4000 × g for 5 minutes at 4°C, collect the cells into a centrifuge tube, resuspend the cells with PBS (pH 7.2), centrifuge at 4000 × g for 5 minutes at 4°C, add an appropriate amount of PBS, and adjust the concentration to OD 600 =10.

[0111] (4) Take out 0.5 mL of bacterial suspension as a blank control, and add florfenicol to the remaining bacterial suspension to adjust the final concentration to 16 μg / mL.

[0112] (5) After the addition of florfenicol and the mixture was allowed to stand for 15 min, compound ZINC338037 and efflux pump inhibitor CCCP (mitochondrial oxidative phosphorylation uncoupler) were added to different groups. 0.5 mL of bacterial solution was taken out at 20, 25, and 30 min, respectively, and added to a centrifuge tube containing 2.5 mL of pre-cooled PBS for dilution. The mixture was then centrifuged at 4°C, 6000 × g for 5 min, the supernatant was discarded, and the tube weight before and after bacterial precipitation was measured to obtain the net weight of the bacteria.

[0113] (6) Resuspend the cells in 1.5 mL of deionized pure water and freeze and thaw repeatedly using liquid nitrogen and a 65°C water bath, changing the temperature every 3 min. Lyse the cells and obtain the supernatant by centrifugation at 6000 × g for 15 min.

[0114] (7) The supernatant was centrifuged and filtered, and filtered into a liquid sample bottle using a 0.22 μm filter membrane. Standard florfenicol with concentrations of 16, 8, 4, 2, 1, and 0.5 μg / mL was prepared and similarly filtered and added to the sample bottle.

[0115] (8) Prepare 12% acetonitrile aqueous solution and methanol, filter using an electric filter, 0.22 μm, and finally use ultrasonic cleaning.

[0116] (9) The samples were detected using a liquid chromatograph with an ultraviolet light of 223 mm, an injection volume of 20 μL, and a flow rate of 1 μL / min.

[0117] (10) The concentration of the liquid phase results was calculated according to the standard curve and the results were expressed as: ng (florfenicol) / mg (wet weight of bacteria) and analyzed using the T-tset method.

[0118] The results of the efflux inhibition test were:

[0119] The results are as follows Figure 5 As shown in A, the efflux pump inhibitor CCCP was used as a positive control. After the compound ZINC338037 was added at 15 minutes, the accumulation concentration of florfenicol in the two strains increased over time (20 minutes, 25 minutes, and 30 minutes). After the addition of the compound ZINC338037 and CCCP, the concentration of florfenicol accumulated in the strain NCTC 11168 was significantly increased compared with the blank control group. At 20 minutes and 25 minutes, the accumulation concentration of florfenicol in the compound ZINC338037 group was lower than that in the CCCP group, and at 30 minutes, the accumulation concentration exceeded that in the CCCP group. Figure 5 As shown in Figure B, after adding compounds ZINC338037 and CCCP to 11168-RE-CmeABC, the results were similar to those of NCTC 11168. At 20 min and 25 min, the accumulated concentration of florfenicol in the compound ZINC338037 group was lower than that in the CCCP group. At 30 min, the accumulated concentration of florfenicol in the compound ZINC338037 group was significantly increased.

[0120] In summary, the results of the efflux inhibition test proved that the combination of compound ZINC338037 and florfenicol can increase its accumulation in bacteria and has the effect of reducing drug efflux.

[0121] Example 10 Cytotoxicity Test

[0122] In order to evaluate the safety of ZINC338037, the toxicity of the drug at different concentrations on African green monkey kidney cells (Vero) and pig kidney cells (PK-15) was detected, and florfenicol was used as a control drug. The specific steps are as follows:

[0123] (1) Preparation of compound solution: Compound ZINC338037 was prepared into a solution with a concentration of 25600 μg / mL using DMSO, and then diluted in two-fold with complete culture medium to prepare test solutions of different concentrations, so that the final concentrations were 512, 256, 128, 64, 32, 16, 8, and 4 μg / mL, respectively.

[0124] (2) Determination of cell survival rate: African green monkey kidney cells (Vero) and porcine kidney cells (PK-15) were passaged to the 3rd to 6th generation, and the cells were in a relatively stable state. The cells were digested from the culture dish and counted, and 500 μL of the diluted cell suspension was added to each well of a 24-well plate. The 24-well plate was gently shaken and placed in an incubator. The cells were cultured in the 24-well plate for 18-24 hours, and the compound to be tested was added after the cells adhered to the wall and grew. The compound to be tested was set at different concentrations, and three parallel controls were used for each concentration. After 24 hours, CCK-8 solution was added. After incubation for 4 hours, the OD value was measured at 450 nm on a microplate reader to calculate the cell survival rate.

[0125] The results of the cytotoxicity test were:

[0126] like Figure 6 As shown in A, in Vero cells, when the concentration of compound ZINC338037 was 512 g / mL, the cell survival rate was 100%, which was the same as the cell survival rate of the control group florfenicol; Figure 6 As shown in Figure B, in PK-15 cells, the cell survival rates of compound ZINC338037 at concentrations of 512, 256, 128, and 64 g / mL were 73%, 74%, 77%, and 97%, respectively, and at 32 g / mL, the cell survival rate reached 100%; the cell survival rates of florfenicol at the corresponding concentrations were 71%, 77%, 93%, and 100%, respectively, and the corresponding drug concentrations of 100% cell survival rates of the two differed by 2 times, indicating that its safety is similar to that of florfenicol. Therefore, compound ZINC338037 has a higher safety in terms of cytotoxicity.

[0127] Example 11 Red blood cell hemolysis test

[0128] In order to determine the safety of ZINC338037 in hemolysis, the red blood cell hemolysis test was used for testing, and florfenicol was used as a control drug to test the safety of different concentrations of ZINC338037 on RBC. The specific steps are as follows:

[0129] (1) PBS buffer: PBS phosphate buffer was prepared with ultrapure water to make 1× PBS, and then sterilized by high pressure.

[0130] (2) Preparation of 5% red blood cell suspension: Take 300 μL of blood into a 10 mL EP tube, add 5700 μL of 1×PBS, mix well, and centrifuge at low temperature (4°C, 3500 rpm, 10 min), discard the supernatant, add 5700 μL of 1×PBS, mix well, and centrifuge at low temperature, repeat this operation until the supernatant is colorless. Finally, resuspend the blood cells at the bottom of the EP tube with 5700 μL of 1×PBS. Prepare 0.1% Triton X-100 solution with 1×PBS as a positive control.

[0131] (3) Take 150 μL of 5% red blood cell suspension and place it in a 96-well plate, add 50 μL of compound ZINC338037, specifically: add 640 μg / mL (final concentration) to the first column, add 320 μg / mL to the second column, and so on to the 10th column. Add 50 μL / well of PBS solution to the 11th column as a negative control, and add 50 μL of 0.1% Triton X-100 to the last column as a positive control.

[0132] (4) After being placed at 37°C for 1 hour, centrifuge at 3500 rpm for 10 minutes and take 100 μL of the supernatant into a new 96-well plate. Measure its absorbance at 540 nm and calculate the hemolysis rate of red blood cells. Hemolysis rate (%) = [(OD 待测样品 -OD 阴性 ) / (OD 阳性 -OD 阴性 )]×100%.

[0133] The results of the red blood cell hemolysis test are:

[0134] like Figure 7 As shown, compound ZINC338037 showed slight hemolysis at 640μg / mL and 320μg / mL, both of which were 1.3%. At the same concentration, the hemolysis rate of florfenicol in the control group was 2% and 0.6%. When the drug concentration was 128μg / mL, the hemolysis rates of compound ZINC338037 and florfenicol were both reduced to 0. Therefore, at concentrations of 128μg / mL and lower, compound ZINC338037 has a higher safety of red blood cell hemolysis.

Claims

1. A method for screening inhibitors of Campylobacter multidrug-resistant efflux pump variant RE-CmeABC, characterized in that: The steps include: 1) Based on the RE-CmeB protein sequence in the NCBI database and the SWISS MODLE website, the homology model of the key efflux protein RE-CmeB in RE-CmeABC was predicted and constructed, and then modified, optimized and comprehensively evaluated to obtain the RE-CmeB screening model; 2) docking the multiple antimicrobial drugs that can be effluxed by RE-CmeABC with the screening model obtained in step 1) to determine the specific binding pocket of RE-CmeB; 3) Create a natural product small molecule ligand database based on ZINC15 data, use the screening model binding pocket obtained in step 2) as the receptor, use the software VINA to dock the obtained protein screening model with the small molecules in the natural product database, and then perform drug-like screening, ADMET screening and molecular dynamics simulation to achieve screening of small molecule compounds, and finally screen out compounds that can serve as inhibitors of the functionally enhanced multidrug resistance efflux pump variant RE-CmeABC, and conduct further drug validation tests on them.

2. The screening method according to claim 1, characterized in that In step 1), the RE-CmeB protein sequence is shown as SEQ ID NO.

1.

3. The screening method according to claim 1, characterized in that In step 2), the antibacterial drug is one or more of florfenicol, ciprofloxacin, erythromycin, tetracycline, and gentamicin.

4. The screening method according to claim 1, characterized in that In step 2), the specific binding pocket of RE-CmeB was determined to be 20 key amino acids: SER47, GLN88, LYS90, ARG131, ILE179, SER177, THR129, ASP152, ILE137, ASN181, SER279, ILE292, GLU275, GLN276, MET571, VAL606, LEU608, PHE611, LEU613, and PHE626.

5. The screening method according to claim 1, characterized in that The molecular docking method in step 3) is as follows: first, the energy is calculated based on the structure and force field parameters of the small molecule, then all possible binding sites around the active pocket of the screening model are searched, and a semi-flexible docking method is selected; according to the scoring standard Affinity of the software VINA, -7 kcal / mol is selected as the threshold, small molecule ligands with scores greater than -7 kcal / mol are excluded, and the part with stronger binding force when the score is ≤-7 kcal / mol is selected.

6. The screening method according to claim 1, characterized in that During drug-likeness screening in step 3), the compounds obtained from the virtual screening are subjected to drug-likeness analysis based on Lipinski's five rules, and potential lead compounds obtained through molecular docking are further screened.

7. The screening method according to claim 1, characterized in that In step 3), the ADMET module of the GHDDI-AIDD platform is used to perform ADMET screening on the results of drug-like screening.

8. The screening method according to claim 1, characterized in that Step 3) During the docking process, GBMV was selected as the implicit solvent model, the atomic radius was set to the van der Waals radius, and molecular dynamics simulation was used for affinity ranking.

9. The Campylobacter function-enhanced multidrug-resistant efflux pump variant RE-CmeABC inhibitor obtained by screening according to the method of claims 1-8, characterized in that: The inhibitor is one or a combination of two or more of ZINC338037, ZINC103293168, and ZINC5999026; the structural formula of the inhibitor is:

10. Use of the multidrug-resistant efflux pump variant RE-CmeABC inhibitor according to claim 9 in enhancing the antibacterial activity of multiple antibiotics against Campylobacter containing the multidrug-resistant efflux pump variant RE-CmeABC.

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