Screening target, application and screening method of antibacterial drug

Abstract

The invention relates to the technical field of medicines, and particularly discloses a screening target of an antibacterial drug, application and a screening method, and the screening method comprises the following steps: testing the interaction between a candidate drug and the target; functional verification of the target protein; testing the antibacterial activity of the candidate drugs; and carrying out an in-vitro antibacterial curve test, evaluating the sterilization or antibacterial effect of the candidate drug on different bacteria, and comparing the change of bacterial gene expression before and after the treatment of the candidate drug through omics analysis (transcriptome and proteome analysis). A new direction is provided for research and development of antibacterial drugs by discovering a new antibacterial target, such as Spx and SodA, of the ISP I except for acting on bacterial ribosomes, a new drug without cross resistance with traditional antibiotics can be developed according to the drug design of the new target, the antibacterial effect is improved, the new antibacterial target of the ISP I in the colimycin and the action mechanism of the new antibacterial target are disclosed, and the application of the ISP I in the colimycin in preparation of the antibacterial drugs is promoted. And a new thought and a new method are provided for research and development of antibacterial drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a screening target, application and screening method of an antibacterial drug. Background Art

[0002] In the field of antimicrobial drugs, with the widespread use of antibiotics, the problem of bacterial resistance is becoming increasingly serious, and traditional antimicrobial drugs are gradually losing their effectiveness, which poses a huge challenge to clinical treatment. At present, the commonly used antimicrobial drugs on the market mainly inhibit bacterial growth by interfering with the function of bacterial ribosomes. However, this mechanism of action easily leads to bacterial resistance. Therefore, the development of antimicrobial drugs with new mechanisms of action to address the problem of bacterial resistance has become an important direction in the current research and development of antimicrobial drugs.

[0003] Macrolide antibiotics are a class of antibacterial drugs widely used in clinical practice. They exert their antibacterial effects by inhibiting the function of bacterial ribosomes. However, due to the continuous emergence of bacterial resistance, the antibacterial effect of traditional macrolide antibiotics has gradually weakened. In recent years, researchers have begun to explore new mechanisms of action of macrolide antibiotics in order to discover new antibacterial drugs with stronger antibacterial activity and lower resistance.

[0004] Carrimycin is a new type of 16-membered macrolide antibacterial drug obtained through biotechnology. Its main components include isovalerylspiramycin (ISP) I, II, III, etc. Compared with traditional macrolide antibiotics, carrimycin has enhanced lipophilicity and tissue permeability, prolonged half-life, and new metabolic pathways. Clinical trials have shown that carrimycin has significant antibacterial activity against a variety of Gram-positive bacteria and certain drug-resistant bacteria, with fewer adverse reactions. However, the specific antibacterial mechanism of carrimycin is not yet fully understood, especially whether it has other antibacterial targets besides acting on bacterial ribosomes, which needs further study.

[0005] In view of this, the present invention aims to further study the antibacterial mechanism of carrimycin, especially to explore whether it has other key antibacterial targets besides ribosomes. By revealing these new targets, the present invention will provide theoretical basis and experimental support for the development of antibacterial drugs with new mechanisms of action, thereby promoting the development of the field of antibacterial drug research and development. Summary of the invention

[0006] The purpose of the present invention is to provide a screening target, application and screening method for antibacterial drugs to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The invention discloses an application of Spx and SodA as drug targets in screening antibacterial drugs, wherein Spx and SodA interact with the candidate drug carrimycin ISPI.

[0009] A method for screening antimicrobial drugs comprises the following steps:

[0010] S1. Test the interaction between candidate drugs and targets; use surface plasmon resonance (SPR) technology to detect the binding affinity between candidate drugs and target proteins, and use electrophoretic mobility shift assay (EMSA) experiments to evaluate the effect of candidate drugs on the binding ability of target proteins to DNA;

[0011] S2. Functional verification of target protein: Use in vitro enzyme activity assay (WST-8 method) to evaluate the effect of candidate drugs on target protein function, use protein structure prediction tool (AlphaFold2) to construct a three-dimensional structural model of target protein binding to candidate drugs, and analyze the interaction mechanism between drugs and targets;

[0012] S3. Antibacterial activity testing of candidate drugs; in vitro inhibition curve tests are performed to evaluate the bactericidal or antibacterial effects of candidate drugs on different bacteria, and changes in bacterial gene expression before and after candidate drug treatment are compared through omics analysis (transcriptome and proteome analysis).

[0013] Preferably, the surface plasmon resonance is specifically to dilute the purified target protein with 10mM sodium acetate pH 5.5 to a 50g / mL protein solution, select Biacore 8K (GE Healthcare, Sweden) to fix the purified target protein to a CM5 chip by amino coupling, and record the RU value. Using the single-cycle kinetics method, 1.05×PBS-P+ buffer containing 5% DMSO is used as the Running Buffer, and different compounds of different concentrations are passed through the SelH fixed on the chip, the binding time is 120s, and the change of RU value is recorded. The equilibrium dissociation constant (KD) is calculated using the software in Biacore 8K to further evaluate the binding affinity between the protein and each compound. The purified target protein is diluted with 10mM sodium acetate pH5.5 to a 50g / mL protein solution, and Biacore 8K (GE Healthcare, Sweden) is selected to fix the purified target protein to a CM5 chip by amino coupling, and the RU value is recorded. The single-cycle kinetics method was adopted, and 1.05×PBS-P+ buffer containing 5% DMSO was used as the Running Buffer. Different compounds of different concentrations were passed through the SelH fixed on the chip. The binding time was 120s, and the changes in RU values ​​were recorded. The equilibrium dissociation constant (KD) was calculated using the software in Biacore 8K to evaluate the binding affinity between the protein and each compound.

[0014] Preferably, the working solution in the WST-8 method for in vitro enzyme activity determination is prepared as follows: according to the volume of 160 μl per reaction, an appropriate amount of WST-8 / enzyme working solution is prepared, and 151 μl of SOD detection buffer, 8 μl of WST-8 and 1 μl of enzyme solution are evenly mixed. The prepared WST-8 / enzyme working solution is stored at 4°C or in an ice bath and used on the same day.

[0015] Preferably, the AlphaFold2 modeling method comprises the following steps:

[0016] 1) Create SodA_monomer and Spx_monomer system folders in the AlphaFold2 folder under the user directory of the computing platform of Yang Zhaoyong's research group at the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences;

[0017] 2) Save the amino acid sequences of SodA and Spx as fasta format files and submit them to the system folder;

[0018] 3) Enter the system folder, select the monomer prediction mode and run the graphical interactive command AF_GUI*. AlphaFold2 searches the UniRef90 and Mgnify databases through JackHMMER and HHBlits to complete protein multiple sequence alignment, and uses HHSearch to find templates. Finally, the modeling is completed through data training and filtering processes;

[0019] The model screening and evaluation methods include: 1) The predicted local distance difference test (pLDDT) score is used to evaluate the modeling accuracy; the models returned by AlphaFold2 are arranged from high to low according to the pLDDT score, and a pLDDT score higher than 90 indicates that the model has high reliability; therefore, the model with the highest pLDDT score greater than 90 is selected for subsequent analysis; 2) The final model of AlphaFold2 is completed using the model quality evaluation module in the online server SWISS MODEL (https: / / swissmodel.expasy.org / assess).

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The discovery of new antibacterial targets of ISPⅠ other than those acting on bacterial ribosomes, such as Spx and SodA, provides a new direction for the research and development of antibacterial drugs. Drug design targeting new targets can develop new drugs with no cross-resistance to traditional antibiotics and improve the antibacterial effect. At the same time, by precisely targeting specific bacterial biomolecules, the nonspecific damage of drugs to host cells can be reduced, and the adverse reaction rate can be reduced.

[0022] The methods and results of this study provide useful reference and lessons for the research and development of other antimicrobial drugs, and help promote innovation and development in the field of antimicrobial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the inhibition curve of macrolide antibiotics such as ISPI of the present invention;

[0024] Figure 2 This is a schematic diagram of SDS-PAGE detection of 8 target proteins in the present invention;

[0025] Figure 3 Schematic diagram of the SPR binding experiment between the target protein and ISPⅠ of the present invention;

[0026] Figure 4 This is a schematic diagram of the EMSA detection of the effect of ISPI on the binding between Spx and the sodA promoter region of the present invention;

[0027] Figure 5 Schematic diagram of the inhibitory effect of SPI and ISP on SodA enzyme activity of the present invention;

[0028] Figure 6 It is a schematic diagram of the three-dimensional structure of the ISPI of the present invention combined with SodA and Spx. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Embodiment 1:

[0031] See also Figures 1 to 6 As shown,

[0032] A method for screening antimicrobial drugs comprises the following steps:

[0033] Experimental material preparation

[0034] Vectors and strains

[0035] pET-His is an expression vector in Escherichia coli and is stored in this laboratory. E. coli DH5a was used for plasmid DNA cloning and is stored in this laboratory; E. coli BL21 DE3 was used as a host bacteria for target gene expression and was purchased from Beijing Quanshijin Biotechnology; Bacillus subtilis 168, Staphylococcus aureus (BNCC186335), Kocuria rhizophila (BNCC336471), and Proteus vulgaris Hauser (BNCC336633) were purchased from Beijing Beina Chuanglian Biotechnology Research Institute.

[0036] Primers

[0037] Table 1 Primers used in this experiment

[0038]

[0039] 2.3 Antibiotics

[0040] Ampicillin (Amp): working concentration is 100 g / mL, from Beijing Dingguo Biotechnology Co., Ltd.

[0041] Common buffer recipes

[0042] (1)NTA-20(1L):

[0043] Tris-HCl 20mM

[0044] NaCl 0.5M

[0045] Imidazole 20 mM

[0046] (2)NTA-500(1L):

[0047] Tris-HCl 20mM

[0048] NaCl 0.5M

[0049] Imidazole 500 mM

[0050] (3) SDS-PAGE gel preparation:

[0051]

[0052] 2.4 Commonly used buffer formulations

[0053] (2)NTA-20(1L):

[0054] Tris-HCl 20mM

[0055] NaCl 0.5M

[0056] Imidazole 20 mM

[0057] (2)NTA-500(1L):

[0058] Tris-HCl 20mM

[0059] NaCl 0.5M

[0060] Imidazole 500 mM

[0061] (3) SDS-PAGE gel preparation:

[0062]

[0063] 2. Experimental Methods

[0064] Antibacterial curve test

[0065] Bacillus subtilis 168 and Staphylococcus aureus (BNCC186335) were cultured in LB at 37°C and 200 rpm overnight. 400 μL of the culture was added to 20 mL of fresh liquid LB medium. The medium was placed in a 250 mL culture flask and cultured at 37°C and 200 rpm until the OD value was about 1. Spiramycin (SP), erythromycin (ERY), azithromycin (AZI) and isovalerylspiramycin I (ISPI) were added at 10 μg / mL each and the culture was continued. The OD600 value was measured every 1 h.

[0066] Omics analysis of Bacillus subtilis 168 after addition of ISPI

[0067] Bacillus subtilis was cultured in LB medium at 37°C until OD600 was approximately white, and ISPI (10 μg / mL) was added. After culturing for another 2 hours, the cells were collected by centrifugation. The control group without ISPI and the sample with ISPI were analyzed for transcriptome and proteome in triplicate, which was commissioned to Shanghai Ouyi Biomedical Technology Co., Ltd.

[0068] Transcriptome analysis

[0069] Total RNA of Bacillus subtilis was extracted by TRIzol (Ambion). After passing the test, rRNA was removed and fragmented, and then reverse transcribed to synthesize cDNA. After connecting the adapter, PCR amplification and purification were performed to construct the PE100 library. After the PE100 library passed the quality inspection, it was sequenced on the machine. High-throughput sequencing was performed using the Illumina HiSeq TM 2000 platform (Shanghai Ouyi). The original sequencing data was first processed by removing the adapter sequence and low-quality sequence to obtain high-quality data, and then compared with the reference gene Bacillus subtilis subsp.168 (AL009126). The transcriptome data were comprehensively evaluated by Tophat2 software for the coverage area and coverage depth of the sequencing data; the gene expression was differentially analyzed by DESeq2 software, and the genes with differential expression between samples were selected by the difference fold (|log2FoldChange|>1) and the significant level (q-value<0.001). The significance threshold of differentially expressed genes in the KEGG metabolic pathway analysis was set to P<0.05.

[0070] Proteome analysis

[0071] (1) Protein extraction

[0072] 1) Take a bacterial cell sample and add an appropriate amount of lysis buffer (8M Urea / 100mM Tris-Cl, pH 8.0) and mix thoroughly.

[0073] 2) Use an ultrasonic disruptor, sonicate for 10 minutes, centrifuge at 12000g for 15 minutes, and take the supernatant.

[0074] 3) The protein in the solution was precipitated using the acetone precipitation method.

[0075] 4) After adding a rehydration solution (8M Urea / 100mM Tris-Cl, pH 8.0) to the obtained protein precipitate for re-dissolution, dithiothreitol (DTT) was added to a final concentration of 10mM, and incubated at 37°C for 1h for reduction reaction to open the disulfide bonds; subsequently, iodoacetamide (IAA) was added to a final concentration of 40mM, and an alkylation reaction was performed at room temperature in the dark to block the thiol groups.

[0076] 5) The protein concentration was determined using the Bradford method.

[0077] 6) After protein quantification, 50 μg of sample was taken for SDS-PAGE and stained with Coomassie Brilliant Blue.

[0078] (2) SDS-PAGE gel electrophoresis

[0079] 50 μg protein was taken from each sample and separated by 12% SDS-PAGE; the separated gel was stained by Coomassie Brilliant Blue staining, referring to the experimental method of Candiano et al. The specific operation is as follows: A. Fix for 2 hours; B. Stain for 12 hours; C. Wash with water until the background is clear; the stained gel was scanned by ImageScanner scanner, the scanning mode is full color mode, and the optical density value is 300dpi.

[0080] (3) Trypsin digestion and labeling

[0081] 1) Add 100 mM Tris-HCl solution (pH 8.0) to the reduced and alkylated samples to dilute the Urea concentration to below 2 M;

[0082] 2) Add trypsin at a mass ratio of 1:50 between enzyme and protein, and incubate at 37°C with shaking overnight for enzyme digestion;

[0083] 3) On the second day, TFA was added to terminate the enzyme cleavage, and the pH value of the solution was adjusted to about 6.0, and centrifuged at 12000g for 15 minutes;

[0084] 4) Take the supernatant and perform Sep-Pak C18 desalting. After the desalted peptide solution is dried by centrifugal concentrator, freeze it at -20℃ for later use.

[0085] 5) Take 100 μg of the enzymatically digested peptides from each sample for iTRAQ labeling. The labeling process was carried out according to the instructions of the iTRAQ manufacturer;

[0086] 6) After the labeled samples were mixed, they were desalted using Sep-Pak C18;

[0087] 7) After vacuum drying, the mixed sample was fractionated using high pH reverse phase chromatography and finally merged into 15 components.

[0088] (4) Chromatographic conditions and mass spectrometry conditions

[0089] 1) Chromatographic conditions

[0090] The peptide samples were aspirated by an autosampler and bound to a C18 capture column (5m, 5x0.3mm), and then eluted to an analytical column (75μmx150mm, 3μm particle size, 100pore size, Eksigent) for separation. Two mobile phases (mobile phase A: 3% DMSO, 97% H2O, 0.1% formic acid and mobile phase B: 3% DMSO, 97% ACN, 0.1% formic acid) were used to establish a 100-min analytical gradient (0min in 5% B, 65min of 5-23% B, 20min of 23-52% B, 1min of 52-80% B, 80% B for 4min, 0.1min of 80-5% B, 5% B for 9.9min). The flow rate of the liquid phase was set at 300nl / min.

[0091] 2) Mass spectrometry conditions

[0092] When the mass spectrometer was analyzed in IDA mode, each scan cycle included a full MS scan (m / z range was 350-1500, ion accumulation time was 250 ms), followed by 40 MS / MS scans (m / z range was 100-1500, ion accumulation time was 50 ms). The MS / MS acquisition conditions were set to a parent ion signal greater than 120 cps and a charge number of +2 to +5. The exclusion time for repeated ion acquisition was set to 18 s.

[0093] Construction of target gene expression plasmids pET-His-traxA, pET-His-perR, pET-His-katA, pET-His-sodA, pET-His-tpx, pET-His-ahpc, pET-His-spx target protein expression strains

[0094] KOD FX NEO enzyme was used to amplify ahpc, kata, tpx, perR, traxA, and spx gene fragments using Bacillus subtilis 168 bacterial culture as a template, and gel electrophoresis experiments and DNA fragment gel recovery were performed; the five gene fragments of ahpc, tpx, perR, traxA, and spx and the pET-His vector were digested with HindⅢ and NdeⅠ, and the katA gene fragment and the pET-His vector were digested with BglII and NdeⅠ, and gel electrophoresis experiments and DNA fragment gel recovery were performed; the recovered gene fragments were then ligated to the pET-His vector using T4 DNA ligase, and the ligated products were transformed into E. coli DH5a, and the transformants selected after Amp resistance were sent to the company for sequencing verification; the E. coli DH5a transformants with correct sequencing were inoculated into 4 mL of fresh LB medium (Amp1005 g / mL) in a shaking tube, cultured overnight at 37°C and 200 rpm, extracted the recombinant plasmid, and then transformed the recombinant plasmid into E. coli BL21 (DE3), and screened the target protein expression strain by Amp resistance.

[0095] Expression and purification of target proteins TrxA, PerR, KatA, SodA, Tpx, Ahpc, Spx

[0096] (1) Take 10 L of the constructed E. coli BL21 (DE3) / pET28a-ahpc, pET28a-kata, pET28a-tpx, pET28a-perR, pET28a-traxA, pET28a-spx target protein expression strains and add them to a shaking tube containing 4 mL of fresh LB medium (Amp: 1005 g / mL) and culture them at 37°C, 200 rpm overnight for activation.

[0097] (2) The activated bacterial solution was transferred to 200 mL of fresh LB medium containing Amp (100 g / mL) resistance at a 1% inoculum volume and cultured at 37°C and 200 rpm until the OD 600 0.6~0.8, add IPTG to a final concentration of 0.2mM (the final concentration of IPTG induction for the target protein in this paper is 0.2mM), and induce at 28℃ or 16℃ for 3-10h.

[0098] (3) Transfer 200 mL of the induced bacterial solution to a 50 mL centrifuge tube, centrifuge at 18,000 rpm for 10 min, collect the bacteria, suspend the bacteria in 10 mL of PBS buffer, disrupt them by ultrasonication, centrifuge at 18,000 rpm for 10 min, and collect the supernatant.

[0099] (4) The target protein was purified using the rapid purification liquid chromatography system AKTA purifier, and impurity proteins were removed using affinity chromatography columns (nickel columns), anion exchange chromatography columns, and gel chromatography columns.

[0100] 1) Nickel column purification: Liquid A is NTA-20, and Liquid B is NTA-500. First, rinse the ethanol in the pipeline and nickel column with deionized water; equilibrate the nickel column with 5 column volumes of NTA-20 and then load the sample (avoid bubbles entering the pipeline when loading the sample); use NTA-20 to flush away the impurities that cannot bind to the nickel column; use Liquid A and Liquid B to gradient elute the target protein and collect it in an EP tube. The flow rate of the entire process is 1 mL / min.

[0101] 2) Anion exchange column purification: Solution A is 20mM Tris, and solution B is 20mM Tris+2M NaCl. This process is similar to the nickel column purification process. Note that if the sample contains imidazole, the buffer in the sample needs to be replaced with PBS buffer using a protein ultrafiltration tube.

[0102] 3) Gel chromatography column purification: Liquid A is PBS buffer. The sample is concentrated to less than 5005L using a protein ultrafiltration tube; first, the ethanol in the pipeline and gel chromatography column is rinsed clean with deionized water; the nickel column is equilibrated with 1 column volume of PBS buffer and then loaded with the sample (air bubbles should be avoided from entering the pipeline during loading); the target protein is eluted with PBS buffer and collected in an EP tube. The flow rate of the entire process is 0.5mL / min.

[0103] (5) The target protein collected in the EP tube is detected using SDS-PAGE experiment.

[0104] EMSA experiments

[0105] Spx protein EMSA experiment: Using the Bacillus subtilis genome as a template, sodA-PF and sodA-PR were used to amplify the promoter region DNA before the open reading frame of the sodA gene. The purified and concentrated Spx protein and ISPI were added to the gel retardation system of the sodA gene promoter region in gradient concentrations and placed at room temperature or shaken for 30 minutes.

[0106] EMSA system:

[0107]

[0108]

[0109] Place the prepared active gel in a vertical electrophoresis tank, use 0.5×TBE as the electrophoresis solution, ice bath, 120V electrophoresis for 30 minutes to remove the effects of impurities in the active gel on the binding of protein and DNA. Replace with a new 0.5×TBE electrophoresis solution, slowly add the reaction solution to the pre-electrophoresis active gel, ice bath, 150V electrophoresis for 90 minutes, carefully remove the gel, place the active gel after electrophoresis in deionized water containing gel-Red nucleic acid dye, shake horizontally for 5 minutes, and place the dyed active gel in a gel imager to observe the results.

[0110] Surface Plasmon Resonance (SPR) was used to identify the binding of colimycin components to target proteins

[0111] Using SelH as a positive control, the SPR method was used to detect the interaction between small molecules and proteins. The purified target protein was diluted with 10mM sodium acetate pH 5.5 to a 50g / mL protein solution. Biacore 8K (GE Healthcare, Sweden) was used to immobilize the purified target protein on the CM5 chip using the amino coupling method, and the RU value was recorded. Using the single-cycle kinetics method, 1.05×PBS-P+ buffer containing 5% DMSO was used as the Running Buffer. Different compounds of different concentrations flowed through the SelH fixed on the chip. The binding time was 120s, and the changes in RU values ​​were recorded. The equilibrium dissociation constant (KD) was calculated using the software in Biacore 8K to evaluate the binding affinity between the protein and each compound.

[0112] Total SOD Activity Detection Kit (WST-8 Method)

[0113] (1) Preparation of WST-8 / enzyme working solution: Prepare an appropriate amount of WST-8 / enzyme working solution according to the volume of 160μl for each reaction. Evenly mix 151μl SOD detection buffer, 8μl WST-8 and 1μl enzyme solution to prepare 160μl WST-8 / enzyme working solution. According to the number of samples to be tested (including standards), prepare an appropriate amount of WST-8 / enzyme working solution. For specific preparation methods, please refer to the table below. The prepared WST-8 / enzyme working solution can be stored at 4℃ or in an ice bath and can be used on the same day, but it is recommended to prepare it as soon as possible.

[0114] (2) Preparation of reaction start working solution: Dissolve the reaction start working solution (40X) in the kit and mix well. Dilute it by adding 39μl SOD detection buffer to every 1μl reaction start working solution (40X). Mix well to obtain the reaction start working solution. Prepare an appropriate amount of reaction start working solution according to the number of samples to be tested (including standards). The prepared reaction start working solution can be stored at 4℃ or in an ice bath and can be used on the same day, but it is recommended to prepare it as soon as possible.

[0115] (3) Preparation of SOD standard: You need to prepare your own SOD standard. Use the SOD sample preparation solution provided by this kit (when the sample is prepared using the SOD sample preparation solution provided by the kit) or SOD detection buffer (when the sample is a sample that does not require treatment, such as blood) to dilute the SOD standard to the following concentration series: 100U / ml, 50U / ml, 20U / ml, 10U / ml, 5U / ml, 2U / ml, 1U / ml. In subsequent tests, 20 microliters can be taken for each reference sample for testing.

[0116] (4) Refer to the table below to set up sample wells and various blank control wells using a 96-well plate. Add the sample to be tested and other solutions in sequence according to the table below. Add the reaction start working solution and mix thoroughly. Note: The reaction will start after adding the reaction start working solution. You can operate at low temperature or use a spray gun to reduce the error caused by the difference in the time of adding the reaction start working solution between each well.

[0117]

[0118] Incubate at 37℃ for 30 minutes. Note: There is no significant difference in SOD activity detected by incubation for 25 to 35 minutes, but in order to ensure the consistency of the test results, it is recommended to incubate for 30 minutes. Measure the absorbance at 450nm. If there is no 450nm filter, a 420-480nm filter can be used. You can choose to set 600nm (or above 600nm, such as 650nm) as the reference wavelength (also called reference wavelength), and the absorbance reading at 450nm minus the absorbance reading at the reference wavelength can be used as the actual measured reading.

[0119] (5) Calculation of total SOD activity in samples:

[0120] a. Calculation of inhibition percentage:

[0121] Refer to the following calculation formula to calculate the inhibition percentage:

[0122] Inhibition percentage = [(A blank control 1 - A blank control 2) - (A sample - A blank control 3)] / (A blank control 1 - A blank control 2) × 100%

[0123] If the sample has no color and does not contain antioxidants, then A blank control 2 = A blank control 3. At this time, the calculation formula can be simplified to the following form (blank control 3 can be omitted during simplification):

[0124] Inhibition percentage = (A blank control 1 - A sample) / (A blank control 1 - A blank control 2) × 100%

[0125] If the calculated inhibition percentage is less than 30% or greater than 70%, the sample usually needs to be re-measured. Try to keep the inhibition percentage of the sample within the range of 30-70%. If the inhibition percentage measured is too high, the sample needs to be appropriately diluted; if the inhibition percentage measured is too low, a higher concentration of the sample needs to be re-prepared.

[0126] b. Definition of SOD enzyme activity unit: When the inhibition percentage in the above xanthine oxidase coupled reaction system is 50%, the SOD enzyme activity in the reaction system is defined as one enzyme activity unit. Note: There are many ways to define the SOD activity unit, and different activity units need to be appropriately converted according to their different definitions.

[0127] AlphaFold2 Modeling

[0128] Modeling: 1) Create SodA_monomer and Spx_monomer system folders in the AlphaFold2 folder under the user directory of the computing platform of Yang Zhaoyong's research group at the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences; 2) Save the amino acid sequences of SodA and Spx as fasta format files and submit them to the system folder; 3) Enter the system folder, select the monomer prediction mode and run the graphical interactive command AF_GUI*. AlphaFold2 completes protein multiple sequence alignment by searching UniRef90 and Mgnify databases through JackHMMER and HHBlits, and uses HHSearch to find templates. Finally, modeling is completed through data training and filtering.

[0129] Model screening and evaluation: 1) The predicted local distance difference test (pLDDT) score was used to evaluate the modeling accuracy. The models returned by AlphaFold2 were arranged from high to low according to the pLDDT score. A pLDDT score higher than 90 indicates high model reliability. Therefore, the model with the highest pLDDT score greater than 90 was selected for subsequent analysis. 2) The final model of AlphaFold2 was completed using the model quality assessment module in the online server SWISS MODEL (https: / / swissmodel.expasy.org / assess).

[0130] Molecular docking

[0131] 1) Small molecule ligand preparation: ISPI was generated by ChemBio3D Ultra 4.0. The root mean square (RMS) gradient was then set to 0.01, and the generated small molecules were energy minimized using the MM2 force field. The file was saved in pdb format for subsequent docking operations.

[0132] 2) Protein file preparation: The SodA and Spx models constructed by AlphaFold2 were loaded into MOE2015.10, and the "compute--repare--QuickPrep" module was used to correct, add hydrogen, and add charge to the SodA and Spx protein structures. The RMS gradient was then set to 0.01, and the system was energy minimized and optimized under the Amber10:EHT force field.

[0133] 3) Run docking: Use the "Site Finder" module to find the active site for docking, and set the highest PLB score item to Dummy Atoms. Then in the "Dock--General" module, select Site as Dummy Atoms. Set the Placement method to Triangle Matcher and the scoring algorithm to London DG; set the Refinement method to Inducedfit and the scoring algorithm to GBVI / WSA dG. After setting the parameters, run the docking software.

[0134] Molecular dynamics simulation

[0135] Molecular dynamics simulations were performed using Gromacs 5.1.5. CHARMM General ForceField (CCenFF) and CHARMM36m force fields were used to construct topological files for small molecule ligands and protein structures, respectively. Visualization results were displayed using pymol and VMD1.9.3.

[0136] Experimental Results

[0137] Test of ISPI antibacterial curve of carrimycin components

[0138] The antibacterial curve test showed that compared with the antibacterial activity of erythromycin (Ery), azithromycin (Azi) and spiramycin (SP), the ISPⅠ component of carrimycin had a significant bactericidal effect against Bacillus subtilis (Bacillus subtilis), Staphylococcus aureus (Staphylococcus aureus, BNCC186335), Kocuria rhizophila (Kocuria rhizophila, BNCC336471), and Proteus vulgaris Hauser (Proteus vulgaris Hauser, BNCC336633) ( Figure 1 A and 1B), but had no antibacterial activity against Gram-negative Escherichia coli DH5a. At a concentration of 10 g / mL, ISPI had a significant bactericidal effect on coccus aureus and Bacillus subtilis, and at a concentration of 0.78

[0139] g / mL can significantly reduce the number of Bacillus subtilis, and as the concentration of ISPI increases, the bactericidal effect becomes more obvious (( Figure 1 C and 1D), indicating that ISPⅠ may have other antibacterial targets besides acting on bacterial ribosomes.

[0140] Omics analysis

[0141] The experimental data were analyzed using ProteinPilot (V4.5) software, and the database used was from the proteome reference database of Bacillus subtilis (Uniprot.Proteome.Bacillus_subtilis.20181109.fasta).

[0142] From the analysis of transcriptome and proteome, it was found that compared with spiramycin, the addition of ISPI affected the differential expression of multiple biological functional genes in B. subtilis, especially the expression of multiple antiperoxidases, including PerR (peroxide operon regulator), KatA (Vegetative catalase 1), Spx (ArsRfamily transcriptional regulator), TrxA (thioredoxin), SodA (superoxidedismutase), AhpC (alkyl hydroperoxide reductase), etc. Among them, the transcription level of perR increased significantly, while the protein level decreased by 2.4 times; the transcription levels of kataA, ahpC, and spx, which were negatively regulated by the transcription level of PerR, increased, while the protein level did not change significantly; and the transcription levels and protein levels of sodA, tpx, and trxA, which were regulated by Spx, decreased. Therefore, we focused on studying whether there is a direct interaction between TrxA, PerR, KatA, SodA, Tpx, Ahpc and Spx proteins and ISPI in carrimycin. First, these proteins were expressed and purified.

[0143] Table 2 Genes with large expression differences in Bacillus subtilis affected by ISPI in omics data

[0144]

[0145]

[0146] Expression and purification of target proteins TrxA, PerR, KatA, SodA, Tpx, Ahpc, Spx

[0147] The molecular weights of target proteins TrxA, PerR, KatA, SodA, Tpx, Ahpc, and Spx are 18kD, 16kD, 54kD, 23kD, 16kD, 21kD, and 16kD, respectively. SelH is a positive protein with a size of 14kD. These target proteins were induced to express soluble proteins, and impurities were removed by nickel column, anion exchange column, and gel column. From the SDS-PAGE test, target proteins of corresponding sizes were obtained, and the purity of the obtained target proteins was high (see Figure 2 ).

[0148] Target protein and carlimycin binding experiment

[0149] Carrimycin is a multi-component drug, and ISPI is one of its main components. For the convenience of research, we studied the interaction between ISPI and various proteins through SPR. As shown in the figure, ISPI has a strong binding effect with the control protein SelH. Among the other 7 antioxidant target proteins, SodA and Spx have a strong binding effect with ISP, and the other proteins have a weak binding effect with ISPI. (See Figure 3 ).

[0150] Spx is a transcriptional regulatory factor that regulates the genes of sodA, tpx, and trxA. Figure 4 It can be seen that Spx binds to the promoter DNA of sodA in a dose-dependent manner. After adding 2.5 g / mL of isopentyl I, the free sodA promoter DNA increased significantly, indicating that isopentyl I affects the binding of Spx to the sodA promoter.

[0151] ISPI affects the enzymatic activity of SodA

[0152] The enzyme activity of SodA was determined by WST-8 method. Figure 5 It can be seen that compared with SPI, ISPI has a significant inhibitory effect on the enzyme activity of SodA, indicating that ISPI affects the enzyme activity of SodA.

[0153] Alphafold simulates the three-dimensional structure of ISPI binding to SodA and Spx

[0154] like Figure 6 The three-dimensional structure of ISPI binding to SodA and Spx

[0155] The three-dimensional structure of ISPI binding to SodA and Spx was evaluated by AlphaFold2, and the binding ΔG of ISPI and SodA docking binding The binding ΔG of ISPI and Spx docking is -7.4 kcal / mol. binding The value of -6.8 kcal / mol indicates that ISPI has a strong binding effect with SodA and Spx. Glu55, Lys83, His79, Glu7 and Glu4 on SodA interact with ISPI, and Glu55 has a hydrogen bond interaction with isovaleryl. Spx interacts with Arg91, Arg92, Ser12 and Thr11. The three-dimensional structure of ISPI binding to SodA and Spx also confirms that ISPI has a strong binding effect with these two proteins.

[0156] In summary, ISPI in chloramphenicol can bind to Spx and SodA in the peroxide scavenging enzyme system. After ISPI binds to Spx, it can affect the transcriptional activity of SodA, and Spx may also affect other peroxide scavenging enzyme genes regulated by it, such as trxA and tpx. After ISPI binds to SodA, it will significantly inhibit the enzymatic activity of its peroxidase. Therefore, in addition to binding to the bacterial ribosome, ISPI in chloramphenicol is likely to affect the functions of Spx and SodA, thereby increasing the peroxide in the bacteria, destroying bacterial macromolecules such as proteins and nucleic acids, and causing bacterial death. Spx and SodA can be used as new antibacterial targets for in-depth research, and the two can be used to screen other inhibitors as antibacterial candidates.

[0157] It will be appreciated that in the development of any actual implementation, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort may be complex and time-consuming, but will be a routine task of design, fabrication, and production for those of ordinary skill having the benefit of this disclosure without undue experimentation.

[0158] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Application of Spx and SodA as drug targets in screening antibacterial drugs, wherein Spx and SodA interact with the candidate drug ingredient carrimycin ISPI.

2. A method for screening antimicrobial drugs, characterized in that: The following steps are involved: S1. Test the interaction between candidate drugs and targets; use surface plasmon resonance (SPR) technology to detect the binding affinity between candidate drugs and target proteins, and use electrophoretic mobility shift assay (EMSA) experiments to evaluate the effect of candidate drugs on the binding ability of target proteins to DNA; S2. Functional verification of target protein: Use in vitro enzyme activity assay (WST-8 method) to evaluate the effect of candidate drugs on target protein function, use protein structure prediction tool (AlphaFold2) to construct a three-dimensional structural model of target protein binding to candidate drugs, and analyze the interaction mechanism between drugs and targets; S3. Antibacterial activity testing of candidate drugs; conduct in vitro inhibition curve tests to evaluate the bactericidal or antibacterial effects of candidate drugs on different bacteria, and compare the changes in bacterial gene expression before and after candidate drug treatment through omics analysis (transcriptome and proteome analysis).

3. The method for screening an antibacterial drug according to claim 1, characterized in that: The EMSA experiment specifically uses the Bacillus subtilis genome as a template, uses sodA-PF and sodA-PR to amplify the promoter region DNA before the open reading frame of the sodA gene, adds the purified and concentrated Spx protein and ISPI to the gel retardation system of the sodA gene promoter region according to gradient concentrations, and leaves it at room temperature or shakes it for 30 minutes. The prepared active gel is loaded into a vertical electrophoresis tank, 0.5×TBE is used as an electrophoresis liquid, and an ice bath is performed at 120V for 30 minutes to remove the influence of impurities in the active gel on the binding of protein and DNA, and a new 0.5×TBE electrophoresis liquid is replaced, and the reaction solution is slowly added to the pre-electrophoresis active gel, and an ice bath is performed at 150V for 90 minutes. The gel is carefully disassembled, and the active gel after electrophoresis is placed in deionized water containing gel-Red nucleic acid dye, and shaken horizontally for 5 minutes. The stained active gel is placed in a gel imager to observe the results.

4. The method for screening an antibacterial drug according to claim 1, characterized in that: The surface plasmon resonance is specifically performed by diluting the purified target protein with 10 mM sodium acetate pH 5.5 into a 50 μg / mL protein solution, using Biacore 8K (GE Healthcare, Sweden) to immobilize the purified target protein on a CM5 chip by an amino coupling method, recording the RU value, using a single-cycle kinetics method, using 1.05×PBS-P+ buffer containing 5% DMSO as a Running Buffer, flowing different compounds of different concentrations through the SelH immobilized on the chip, the binding time is 120 s, and recording the change in RU value; using the software in Biacore 8K to calculate the equilibrium dissociation constant (KD), and then evaluating the binding affinity between the protein and each compound. The purified target protein is diluted with 10 mM sodium acetate pH 5.5 to a 50 μg / mL protein solution, using Biacore 8K (GE Healthcare, Sweden) to immobilize the purified target protein on a CM5 chip by an amino coupling method, and recording the RU value; using a single-cycle kinetics method, using 1.05×PBS-P+ buffer containing 5% DMSO as a Running Buffer, flowing different compounds of different concentrations through the SelH immobilized on the chip, the binding time is 120 s, and recording the change in RU value. The kinetics method was used, using 1.05×PBS-P+ buffer containing 5% DMSO as the Running Buffer. Different compounds of different concentrations were passed through the SelH fixed on the chip. The binding time was 120 s, and the changes in RU values ​​were recorded. The equilibrium dissociation constant (KD) was calculated using the software in Biacore 8K to evaluate the binding affinity between the protein and each compound.

5. The method for screening an antibacterial drug according to claim 1, characterized in that: Preparation of working solution in the WST-8 method for in vitro enzyme activity determination: prepare an appropriate amount of WST-8 / enzyme working solution according to the volume of 160 μl per reaction, and evenly mix 151 μl SOD detection buffer, 8 μl WST-8 and 1 μl enzyme solution; store the prepared WST-8 / enzyme working solution at 4°C or in an ice bath and use it on the same day.

6. The method for screening an antibacterial drug according to claim 1, characterized in that: The AlphaFold2 modeling method includes the following steps: 1) Create SodA_monomer and Spx_monomer system folders in the AlphaFold2 folder under the user directory of the computing platform of Yang Zhaoyong's research group at the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences; 2) Save the amino acid sequences of SodA and Spx as fasta format files and submit them to the system folder; 3) Enter the system folder, select the monomer prediction mode and run the graphical interactive command AF_GUI*. AlphaFold2 searches the UniRef90 and Mgnify databases through JackHMMER and HHBlits to complete protein multiple sequence alignment, and uses HHSearch to find templates; finally, the modeling is completed through data training and filtering processes; The model screening and evaluation methods include: 1) The predicted local distance difference test (pLDDT) score is used to evaluate the modeling accuracy; the models returned by AlphaFold2 are arranged from high to low according to the pLDDT score, and a pLDDT score higher than 90 indicates high model reliability; therefore, the model with the highest pLDDT score greater than 90 is selected for subsequent analysis; 2) The final model of AlphaFold2 is calculated using the online server SWISS MODEL The model quality assessment module in (https: / / swissmodel.expasy.org / assess) is completed.

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