Application of enrofloxacin in the preparation of antibacterial agents for drug-resistant bacteria and in the preparation of drugs for treating tuberculosis

Abstract

The present invention proposes an application of enrofloxacin in preparing an antibacterial agent for drug-resistant bacteria and an application of enrofloxacin in preparing a drug for treating tuberculosis, and relates to the field of biotechnology. The present invention proposes an application of enrofloxacin in preparing an antibacterial agent for inhibiting drug-resistant Mycobacterium tuberculosis and an application of enrofloxacin in preparing a drug for treating diseases caused by drug-resistant Mycobacterium tuberculosis. The technical solution of the present invention can reduce the activity of drug-resistant bacteria in an in vitro environment by using enrofloxacin as an inhibitor for inhibiting drug-resistant bacteria. In particular, enrofloxacin, as a quinolone derivative, can specifically inhibit the growth and development of Mycobacterium tuberculosis through the GyraseAB target.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of enrofloxacin in preparing a bacteriostatic agent for drug-resistant bacteria and a medicine for treating tuberculosis. Background Art

[0002] Tuberculosis (TB) is a highly pathogenic infectious disease caused by infection with Mycobacterium tuberculosis (Mtb). It is the disease with the highest mortality rate caused by a single infectious source. At present, the treatment of tuberculosis mainly adopts the DOTS regimen recommended by WHO, and the treatment cycle is as long as half a year. Due to the long treatment cycle, poor patient compliance and the unique physiological characteristics of Mtb, tuberculosis has a serious problem of drug resistance. Compared with the previous year, the number of patients with drug-resistant tuberculosis has also increased significantly. In recent years, total drug-resistant (TDR) strains that are resistant to almost all antibiotics have also appeared. Traditional anti-tuberculosis drugs cannot effectively treat tuberculosis caused by drug-resistant Mtb infection, and its cure rate is only 55%. However, the development of anti-tuberculosis drugs has been slow. In the past fifty years, only three anti-tuberculosis drugs with new structures, Bedaquiline, Delamanid and PA-824, have been approved for the treatment of (drug-resistant) tuberculosis. However, these new drugs are highly toxic, and Mtb strains resistant to them have quickly emerged in the clinic. Therefore, it is urgent to develop new anti-tuberculosis drugs for the treatment of tuberculosis caused by drug-resistant Mtb infection.

[0003] In recent years, a high-throughput inhibitor screening model at the molecular level has been established based on the products encoded by essential genes, and a large number of compounds with enzyme inhibitory activity have been obtained. However, the shielding effect of the cell wall and cell membrane of Mtb and the complex metabolic environment inside the cell make them inactive. The establishment of targeted drug screening at the cell level overcomes this obstacle and excludes compounds that cannot enter the cell and reach the target site. Traditional anti-tuberculosis drugs (or their precursors) are obtained in this way, and the precursors of new anti-tuberculosis drugs such as Bedaquiline, Delamanid, PA-824 and linezolid are also discovered through this research method. Therefore, screening studies of inhibitors at the cell level are more likely to obtain inhibitors with development value. Summary of the invention

[0004] The main purpose of the present invention is to propose an application of enrofloxacin in the preparation of a bacteriostatic agent for drug-resistant bacteria and a drug for treating tuberculosis, aiming to solve the problem that existing bacteriostatic agents may cause drug resistance in target strains.

[0005] To achieve the above object, the present invention proposes a use of enrofloxacin in the preparation of an antibacterial agent for drug-resistant bacteria.

[0006] In one embodiment, the drug-resistant bacteria include drug-resistant Mycobacterium tuberculosis.

[0007] In one embodiment, the minimum inhibitory concentration of enrofloxacin is 0.8-10 µM.

[0008] The invention also provides an application of enrofloxacin in preparing medicine for treating tuberculosis.

[0009] The technical solution of the present invention can reduce the activity of drug-resistant bacteria in an in vitro environment by using enrofloxacin as an inhibitor for inhibiting drug-resistant bacteria. In particular, enrofloxacin, as a quinolone derivative, can specifically inhibit the growth and development of Mycobacterium tuberculosis through the GyraseAB target. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0011] Figure 1 The 3D-QSAR model molecular superposition, prediction accuracy fitting relationship, and 3D field force analysis diagram in Example 1 of the present invention; Figure 2 This is a docking diagram of GyraseA+B and DNA with quinolone drug molecules in Example 2 of the present invention; Figure 3 RMSD diagram of each biological molecule and drug molecule in the molecular dynamics system in Example 2 of the present invention; Figure 4 RMSF diagram of each protein in the molecular dynamics system in Example 2 of the present invention; Figure 5 It is a fitting diagram of the 3D-QSAR predicted values ​​and actual values ​​of the 7 compounds tested in Example 4 of the present invention; Figure 6 This is an electrophoretic diagram of the test of the inhibitory effect of levofloxacin and moxifloxacin in the control group compared with enrofloxacin and chloroquine on the desupercoiling function of the Gyrase AB target enzyme in Example 5 of the present invention.

[0012] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings in conjunction with the embodiments. DETAILED DESCRIPTION

[0013] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0014] It should be noted that, in the embodiments, those without specifying specific conditions are carried out according to normal conditions or conditions recommended by the manufacturer. Those without specifying the manufacturer of reagents or instruments used are conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, and "A and / or B" is taken as an example, including schemes A, B, or A and B that meet the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of the technical schemes is contradictory or cannot be achieved, it should be considered that the combination of such technical schemes does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work, all belong to the scope of protection of the present invention.

[0015] In view of this, the present invention proposes a use of enrofloxacin in the preparation of an antibacterial agent for drug-resistant bacteria.

[0016] The technical solution of the present invention adopts the Mtb standard strain H37Rv (ATCC27294) to establish a high-throughput inhibitor screening model at the bacterial level, and screens the compound library L4000 (containing 7285 compounds, original concentration 10 mM) purchased from Shanghai Taosu Biotechnology Co., Ltd. (initial screening concentration 10 μM), and finally screens out the compound enrofloxacin (Enrofloxacin, CAS: 93106-60-6), which shows strong inhibitory activity against both Mtb-sensitive bacteria and clinically resistant bacteria, and has great development prospects.

[0017] The technical solution of the present invention can reduce drug-resistant bacteria in an in vitro environment by using enrofloxacin as an inhibitor for inhibiting drug-resistant bacteria. In particular, enrofloxacin, as a quinolone derivative, can specifically inhibit the growth and development of Mycobacterium tuberculosis through the GyraseAB target.

[0018] In one embodiment, the drug-resistant bacteria include drug-resistant Mycobacterium tuberculosis.

[0019] In one embodiment, the minimum inhibitory concentration of enrofloxacin is 0.8-10 µM.

[0020] In one embodiment, the inhibitory ability of enrofloxacin on the target Gyrase AB complex function is slightly lower than that of levofloxacin.

[0021] In one embodiment, the binding affinity of enrofloxacin to the target GyraseAB complex is -29 kcal / mol, which is comparable to the binding affinity of levofloxacin and moxifloxacin (-33.63 kcal / mol).

[0022] The invention also provides an application of enrofloxacin in preparing medicine for treating tuberculosis.

[0023] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0024] Experimental Materials Medium 7H9, medium 7H10 and additive OADC were purchased from BD.

[0025] Mtb H37Rv (ATCC 27294), clinical multidrug-resistant strains Mtb 28 and 1731 were maintained by the Bacterial Immunology Department of Beijing Chest Hospital, Capital Medical University & Beijing Institute of Tuberculosis and Thoracic Tumors.

[0026] Mycobacterium smegmatis Mycolicibacterium smegmatis (Msm) mc2155, Mycobacterium marinum Mycobacterium marinum (Mma) BAA535, Mycobacterium neoaurea Mycobacterium neoaurum (Mne) 25795 is deposited in the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences.

[0027] Quinolone inhibitors, including chloroquinaldol (CID 72-80-0), enrofloxacin (CID 93106-60-6), 5-chloro-8-hydroxyquinoline (CID 130-16-5), lenvatinib (CID 417716-92-8), 5-chloro-7-(pyrrolidin-1-ylmethyl)-8-hydroxyquinoline (CID F0870-0079), 2,6-dimethyl-4-(3,4-dimethylamino)quinoline (CID 4491-0991), and 5-chloro-7-((4-phenylmethylpiperidin-1-yl)methyl)-8-hydroxyquinoline (CID F0743-0024), were purchased from Shanghai Taosu Biotechnology Co., Ltd.

[0028] A supercomputing server: CPU: Intel9900K, memory 64GB, GPU: 4090Ti Ubuntu22.

[0029] pET-21a, pET-N-His-TEV plasmids, BL21 (DE3) pLysS expression system, supercoiled pBR322 plasmid, Tris-HCl buffer, HEPES.KOH buffer.

[0030] Example 1 The force field-based 3D-QSAR model was constructed using Schrödinger 2022-1 software.

[0031] First, 149 molecules were optimized (LigPrep), and OPLS was used as the force field. Each molecule was optimized for 500 steps. When modeling, the default 3D descriptors of the molecules were used: energy (kcal / mol), charge optimization score, ion score, and ADMET score. The parameters were adjusted to Gaussian force field and 10 groups of partial least squares factors (PLS factors).

[0032] The results are as follows Figure 1 As shown, A is the molecular superposition diagram of the 3D-QSAR model; B is the fitting relationship of the training set of the best 3D-QSAR model, where R 2 =0.83, and the model has good predictive ability, Q 2 =0.59; Figure C is a 3D field force analysis diagram.

[0033] After using this QSAR model to predict 7 quinolone inhibitors, it was found that enrofloxacin ranked second and chloroquinedol ranked first.

[0034] Example 2 Based on the 3D QSAR model, enrofloxacin and chloroquinaldol were selected, and the two molecules were modeled and optimized. Since the main target of quinolone drugs is GyraseB, and previous studies have found that quinolones bind to the GyraseA-GyraseB-DNA complex. Therefore, we used Schrödinger 2022-1's Glide-dock docking software to dock them into the corresponding target complex: GyraseA-GryaseB-DNA. The docking scores of the two (enrofloxacin is -12.2 kcal / mol and chloroquinaldol is -9.5 kcal / mol) are both within the acceptable range.

[0035] like Figure 2 As shown, the left figure is a thumbnail of GyraseA+B, DNA and inhibitor molecules, and the right figure is a comparison of the pdb structures of levofloxacin and moxifloxacin with the docking structure of enrofloxacin. In the right figure, the re-docking scores of levofloxacin, moxifloxacin and enrofloxacin and the docking scores of quinolones are shown in the lower left corner.

[0036] The quinolone skeleton of chlorquinaldol and enrofloxacin inhibitors is consistent with the superposition of the levofloxacin and moxifloxacin in the X-ray crystal structure.

[0037] Example 3 The Amber molecular dynamics simulations of the complexes of these drugs and inhibitors (including levofloxacin, moxifloxacin, norfloxacin and chloroquine) with the target were performed for 200 ns and repeated three times for each system (e.g. Figure 3 The RMSD indicator and Figure 4 The results show that enrofloxacin is more stably bound in the drug cavity, similar to levofloxacin and moxifloxacin. In the simulation, enrofloxacin forms a pi-pi Interaction. Chloroquinaldol easily moves between different bases, and has the potential to be released from the binding cavity and cause off-target. Secondly, we calculated the binding free energy of the four drugs in the kinetic simulation by MM-GBSA and MM-PBSA. As shown in Table 1, the binding free energy of enrofloxacin and the receptor complex (enrofloxacin: G gbsa = -29 kcal / mol, G pbsa = -8.63 kcal / mol) and the binding free energy of levofloxacin to the receptor complex (G gbsa = -33.63 kcal / mol, G pbsa = -1.44 kcal / mol), and chloroquinetolide was even found in PBSA calculations to be unsuitable for binding to the binding domain of the target complex (G pbsa = 4.98 kcal / mol), so computational biology theory proves that the target of enrofloxacin is the target complex of the pathogen: GyraseA-GryaseB-DNA. Based on this theoretical simulation, it is speculated that enrofloxacin should have potential anti-tuberculosis activity.

[0038] Table 1 Binding free energy results of drug molecules in various systems in Example 3

[0039] Example 4 Use an inoculation loop to dip into Mtb H37Rv and clinical multidrug-resistant strains Mtb28 and Mtb1731 stored at -80℃ and streak on 7H10 solid medium (containing 10% OADC), incubate at 37℃ until a single colony grows, pick a single colony and inoculate it in 5mL 7H9 medium (containing 10% OADC and 0.05% Tween80), and incubate it for 10-14 days. In the same way, activate strains Msm (37℃, growth cycle 1-3 days), Mne (37℃, overnight culture) and Mma (28℃, growth cycle 5-7 days).

[0040] The MIC values ​​of the inhibitors obtained in 1.2.3 against Mtb H37Rv and clinically isolated MDR strains Mtb28 and Mtb1731 were determined in 96-well plates using the two-fold dilution method. 200 µL of 7H9 medium was added to rows 1 and 8 to prevent drying. The highest test concentration was 10 µM and the lowest was 0.08 µM (3 parallels). The solvent DMSO was used as a negative control and INH was used as a positive control. In these samples, the compound enrofloxacin showed a strong inhibitory effect on both MTB standard strains and clinical strains.

[0041] We conducted a minimum inhibitory concentration (MIC) experiment on seven quinolone inhibitors (including chloroquine and enrofloxacin) selected from our own sample library. The pMIC results of this experiment and the R of the predicted results of the 3D model are 2 The fit is 0.86 (such as Figure 5 The results are shown in Table 2). This indicates that the theoretical pMIC value is similar to the experimental pMIC value. The best quinolone derivative molecules, chloroquinaldol and enrofloxacin, both have tuberculosis inhibitory activity.

[0042] Table 2 MIC experimental results of 7 quinolone inhibitors in Example 4

[0043] Example 5 To test the effects of chlorquinaldol and enrofloxacin on DNA cleavage enzyme activity, we prepared a complex of GyraseA and GyraseB of MTB H37rv bacteria. Under normal circumstances, the GyraseA-GyraseB heterotetramer converts supercoiled DNA (pBR322) into relaxed DNA. In drug experiments, with the addition of small molecule drugs (levofloxacin, moxifloxacin, etc.), molecules with inhibitory activity will promote the tetramer bound to supercoiled DNA to become a linear DNA cleavage complex. When the concentration increases further, all DNA bands will weaken due to the increase in active drug molecules. Our 20 μL reaction system includes: 5× cleavage buffer (50 mM HEPES-KOH [pH 7.9], 100 mM potassium glutamate, 6 mM magnesium acetate, 2 mM spermidine, 0.1 mg / mL albumin, 4 mM DTT), GyraseAB heterotetramer, 0.4 μg supercoiled DNA (pBR322) and a series of quinolone concentrations. After incubation at 37°C for 30 minutes, proteinase K and 2% SDS were added and incubated for another 30 minutes to terminate the reaction.

[0044] In this experimental result, Figure 6 As shown, the maximum half-inhibitory concentration of enrofloxacin on the target complex is between 10-20μM. It is about 10 times higher than moxifloxacin (about 1μM) and 2.5 times higher than levofloxacin (about 5μM). This shows that enrofloxacin has an inhibitory effect on the target complex, and the target complex may be its potential target. Chloroquinaldol has no obvious effect on the activity of the target complex, indicating that the antibacterial activity of chloroquinaldol may come from acting on other tuberculosis target proteins.

[0045] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification under the inventive concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. Use of enrofloxacin in the preparation of an antibacterial agent for drug-resistant bacteria; The drug-resistant bacteria include drug-resistant Mycobacterium tuberculosis; The minimum inhibitory concentration of enrofloxacin is 0.8~10 μM.

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