Pharmaceutical composition or vaccine for treating and / or inhibiting salmonella infection and application of small molecule compound Epetrabole hydrochloride

Abstract

The invention discloses a pharmaceutical composition or a vaccine for treating and / or inhibiting salmonella infection, and an application of a small molecule compound Epetrabole hydrochloride. The invention further discloses an application of the pharmaceutical composition or the vaccine and the small molecule compound Epetrabole hydrochloride for treating and / or inhibiting salmonella infection. The MIC value of the small molecule compound is 0.48 [mu] M, and compared with existing antibiotics for clinically treating salmonella enteritidis infection, the small molecule compound has relatively strong antibacterial activity when the small molecule compound is in a trace amount. An in-vitro killing bacteriostatic activity experiment result shows that the small molecule compound has a certain effect on reduction of the number of bacteria. The Salmonella enteritidis strain has obvious antibacterial activity on a J774A.1 cell model, and can inhibit the expression of a salmonella enteritidis virulence factor T3SS1 in a targeted manner. According to the present invention, the small molecule compound Epetrabole hydrochloride provides a certain inhibition effect on salmonella isolates from different sources, and has a wide application value;

Description

Technical Field

[0001] The present invention relates to the application of a pharmaceutical composition or vaccine for treating and / or inhibiting Salmonella infection and a small molecule compound, Epetraborole hydrochloride, and belongs to the field of biotechnology. Background Art

[0002] Salmonella enteritidis is a common zoonotic pathogen and one of the main pathogenic bacteria causing food poisoning worldwide, often spreading extensively through contaminated water and food. With the continuous increase in drug resistance and the drug resistance transmission mechanism, the abuse and overuse of antibiotics have accelerated the evolution of drug-resistant strains. Addressing microbial drug resistance is a global public health issue. Therefore, developing new drugs to prevent and control Salmonella is an urgent measure.

[0003] Small molecule compounds usually consist of several to dozens of atoms and have a relatively simple molecular structure, which makes their chemical synthesis and structure modification relatively easy to achieve. And in the field of drug research and development, etc., the establishment and screening of small molecule compound libraries are relatively easy and cost-effective. High-throughput screening technology can be used to quickly screen a large number of small molecule compounds to find compounds with specific biological activities, greatly improving the efficiency of discovering new drugs or bioactive molecules. Compared with some biological macromolecules, small molecule compounds usually have better chemical stability and physical stability. They are not easily denatured or degraded by factors such as temperature, pH value, and enzymes, and are more convenient and reliable during storage and use. Based on the above characteristics, small molecule compounds have a wide range of applications in clinical practice and can be used as a brand-new method to effectively prevent and control Salmonella enteritidis. Summary of the Invention

[0004] Object of the Invention: The technical problem to be solved by the present invention is to provide the application of a pharmaceutical composition or vaccine for treating and / or inhibiting Salmonella infection and a small molecule compound, Epetraborole hydrochloride.

[0005] Technical Solution: To solve the above technical problem, the present invention provides a pharmaceutical composition or vaccine for treating and / or inhibiting Salmonella infection, which contains a small molecule drug, Epetraborole hydrochloride.

[0006] Wherein, the Salmonella is Salmonella enteritidis, Salmonella typhimurium, Salmonella london, Salmonella derby, Salmonella kentucky or Salmonella rosen.

[0007] Wherein, the dosage form of the pharmaceutical composition includes tablets, capsules, granules, aerosols, sprays or injections.

[0008] The present invention also provides the use of the small molecule compound Epetraborole hydrochloride in the preparation of a pharmaceutical composition or vaccine for treating and / or preventing Salmonella infection.

[0009] Wherein, the Salmonella is Salmonella enteritidis, Salmonella typhimurium, Salmonella london, Salmonella derby, Salmonella kentucky or Salmonella rosen.

[0010] Wherein, the concentration of the small molecule compound Epetraborole hydrochloride is 0.24 - 4.8 μM.

[0011] Furthermore, the concentration of the small molecule compound Epetraborole hydrochloride is 0.48 - 4.8 μM.

[0012] Furthermore, the concentration of the small molecule compound Epetraborole hydrochloride is 0.96 - 4.8 μM.

[0013] Furthermore, the concentration of the small molecule compound Epetraborole hydrochloride is 2.4 - 4.8 μM.

[0014] Wherein, when the Salmonella is Salmonella enteritidis, the concentration of the small molecule compound Epetraborole hydrochloride is 0.24 - 4.8 μM.

[0015] Wherein, Epetraborole hydrochloride structurally belongs to a small molecule chemical drug, and its molecular structural formula is C 11 H 17 BClNO4, and its chemical name is 3 - [[(3S)-3-(aminomethyl)-1-hydroxy-3H-2,1-benzoxaborol-7-yl]oxy]propan-1-ol; hydrochloride, and its relative molecular mass is 273.52 g / mol. Its structural formula is:

[0016] Wherein, the small molecule compound Epetraborole hydrochloride has safety on J774A.1 cells.

[0017] Among them, the small molecule compound Epetraborole hydrochloride has antibacterial properties on J774A.1 cells and can target and inhibit the virulence factor T3SS1 of Salmonella enteritidis.

[0018] Among them, the small molecule drug Epetraborole hydrochloride is an inhibitor of LeuRS. This drug is mainly used to treat bacterial infections, especially showing great potential in the treatment of multi-drug resistant Gram-negative bacterial infections. By binding to the adenosine ribose at the terminal of leucyl-tRNA synthetase, it inhibits the synthesis of outer membrane proteins of Gram-negative bacteria, thus playing an antibacterial role.

[0019] Among them, the small molecule drug Epetraborole hydrochloride can be used as a control agent for Salmonella enteritidis.

[0020] Among them, the small molecule drug Epetraborole hydrochloride has a certain antibacterial effect on Salmonella of different host origins and different serotypes.

[0021] The small molecule drug Epetraborole hydrochloride that can inhibit the activity of Salmonella enteritidis in the present invention provides a safe and effective drug product for the prevention and control of Salmonella enteritidis. The small molecule drug is mainly used to treat bacterial infections, especially against Salmonella enteritidis. Epetraborole hydrochloride is a novel LeuRS inhibitor that inhibits protein synthesis by binding to the adenosine ribose at the terminal of leucyl-tRNA synthetase and can be used to study infections caused by Gram-negative bacteria. It shows great potential in the treatment of multi-drug resistant Gram-negative bacterial infections.

[0022] Among them, the optimal growth temperature of Salmonella enteritidis is 37°C.

[0023] Among them, the optimal pH of Salmonella enteritidis is 6.5 - 7.5.

[0024] Among them, the optimal salt concentration of Salmonella enteritidis is 0.5 - 1.5% (low salt environment).

[0025] Among them, the incubation period of Salmonella enteritidis is usually 6 - 72 hours, and the specific time depends on the infection dose and host immunity. Typical clinical symptoms are diarrhea, abdominal pain, nausea, vomiting, and fever; systemic symptoms: headache, fatigue, muscle soreness; severe cases may show dehydration.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0027] (1) The small molecule compound Epetraborole hydrochloride of the present invention effectively inhibits various Salmonella serotypes specifically and can be applied to the prevention and control of most Salmonella enteritidis. Moreover, it can achieve a therapeutic effect at a low dose (0.48 μM), aiming to make up for the deficiencies of existing treatment methods and provide a more effective and specific treatment option for patients.

[0028] (2) The small molecule compound Epetraborole hydrochloride has low cytotoxicity in vitro and has good safety.

[0029] (3) The development of this small molecule specific drug not only provides new ideas and methods for the treatment of Salmonella enteritidis infection, but also overcomes many limitations of traditional antibiotic treatment. Its high selectivity, low side effects, low dose, high efficiency and clear mechanism of action make the drug show good application prospects in clinical trials. Description of the Drawings

[0030] Figure 1 Growth curve of Salmonella enteritidis after treatment with different concentrations of Epetraborole hydrochloride;

[0031] Figure 2 Killing activity of the small molecule compound Epetraborole hydrochloride against Salmonella enteritidis;

[0032] Figure 3 Effect of different concentrations of the small molecule compound Epetraborole hydrochloride on the cytotoxicity of J774A.1 cells;

[0033] Figure 4 Bacteriostatic activity of different concentrations of the small molecule compound Epetraborole hydrochloride against Salmonella enteritidis;

[0034] Figure 5 Effect of different concentrations of the small molecule compound Epetraborole hydrochloride on the adhesion and invasion ability of Salmonella enteritidis;

[0035] Figure 6 Determination of the expression level of the T3SS1 gene of Salmonella enteritidis by the small molecule compound Epetraborole hydrochloride at 1 / 5 MIC concentration. Detailed Embodiments

[0036] The technical solutions of the present invention will be further described below in conjunction with the drawings.

[0037] The small molecule compound Epetraborole hydrochloride was purchased from Shanghai TargetMol Biotechnology Co., Ltd.; J774A.1 cells were preserved in the Laboratory of Zoonoses, Yangzhou University, Yangzhou, Jiangsu Province. Other raw materials used, unless otherwise specified, are all ordinary commercially available products and can be obtained through commercial channels.

[0038] Example 1 Screening of Small Molecule Drugs against Salmonella enterica Serovar Enteritidis Strain Z11

[0039] Using the Salmonella enterica Serovar Enteritidis strain Z11 (DOI: 10.1128 / spectrum.01574-22) preserved in the laboratory as the experimental subject, the antibacterial activity of Epetraborole hydrochloride against Salmonella enterica was evaluated. As shown in Table 1, the small molecule drug Epetraborole hydrochloride has the best antibacterial effect on Salmonella enterica Serovar Enteritidis strain Z11, and the MIC value is 0.48 μM.

[0040] Table 1 MIC Values of Small Molecule Compound Epetraborole Hydrochloride against Different Bacteria

[0041]

[0042] Example 2 Growth Curves of Salmonella enterica after Treatment with Different Concentrations of Epetraborole Hydrochloride

[0043] (1) Pick a single colony of Salmonella enterica Serovar Enteritidis strain Z11 and inoculate it into 1 mL of LB liquid medium. Incubate overnight at 37°C with shaking at 180 rpm.

[0044] (2) Expand the culture 1:100 in 1.5 mL of LB liquid medium and incubate statically at 37°C for about 3 h. Centrifuge the bacterial liquid at 5000 rpm for 5 min at 4°C and adjust the OD of the bacterial liquid 600 = 1.

[0045] (3) Dilute the bacterial liquid with LB medium to OD 600 = 0.05, add drugs at concentrations of MIC, 1 / 2MIC, and 1 / 10MIC respectively, and add 200 μL per well to a 96-well bacterial culture plate. Incubate at 37°C with shaking at 180 rpm, measure with an enzyme-linked immunosorbent assay (ELISA) reader every hour for a total of 24 h and analyze the results.

[0046] As Figure 1As shown in the figure, adding Epetraborole hydrochloride at a concentration of MIC (0.48 μM) can effectively inhibit the growth of Salmonella enteritidis Z11. When adding Epetraborole hydrochloride at a concentration of 1 / 2 MIC (0.24 μM), the growth rate of bacteria can be slowed down, and the number of bacteria at the plateau phase is reduced. When adding Epetraborole hydrochloride at a concentration of 1 / 10 MIC (0.048 μM), the growth curve of Salmonella enteritidis is similar to that of the wild strain, with no significant difference. The above results indicate that as the concentration of the small molecule compound Epetraborole hydrochloride increases, it can inhibit the growth of bacteria, and when reaching the MIC concentration, it can completely inhibit the growth of Salmonella enteritidis Z11 strain.

[0047] Example 3 Determination of the killing activity of the small molecule compound Epetraborole hydrochloride against Salmonella enteritidis in vitro

[0048] (1) Pick a single colony of Salmonella enteritidis Z11 and inoculate it into LB liquid medium, and culture it overnight at 37 °C and 180 rpm;

[0049] (2) Expand the culture of the bacterial solution in 2 mL of LB liquid medium, and adjust the initial OD of the bacterial solution after culturing at 37 °C and 180 rpm for 3 h 600 = 1;

[0050] (3) Dilute the small molecule compound Epetraborole hydrochloride with PBS buffer to concentrations of MIC, 1 / 2 MIC, 5 MIC, and 10 MIC respectively, add bacteria and mix well until the final concentration of the bacterial solution is 5×10 5 CFU, immediately perform gradient dilution and plate counting, which is the 0 hour;

[0051] (4) At 3 h and 5 h after adding the bacterial solution, take samples for gradient dilution and plate counting, culture in a 37 °C microbial constant temperature incubator for 10 - 12 h, and calculate the antibacterial activity.

[0052] As Figure 2 shown, as time prolongs, the small molecule compound Epetraborole hydrochloride has different degrees of bactericidal activity against Salmonella enteritidis Z11 strain. At 3 h, the killing effect of the drugs at concentrations of 1 / 2 MIC and MIC on bacteria is not obvious, while at a concentration of 10 MIC, all bacteria can be killed; at 5 h, there are significant differences in the killing effect of drugs at different concentrations on bacteria, and the higher the concentration of the drug, the stronger the killing effect on bacteria.

[0053] Example 4 Evaluation of the safety of the small molecule compound Epetraborole hydrochloride at the cellular level

[0054] (1) Seed J774A.1 cells into a 48-well plate at a density of 1×10 5 cells per well, with a culture medium volume of 300 μL per well, and culture at 37 °C in 5% CO2 until the next day.

[0055] (2) The next morning, add small molecule compounds at concentrations of MIC, 5MIC, and 10MIC respectively, and set up a blank control group. One hour after drug treatment, take out the cell plate, discard the culture supernatant, and replenish with 200 μL of fresh Opti-MEM medium containing gentamicin (final concentration 50 μg / mL), and continue treatment for 3 h.

[0056] (3) Two hours after the end of treatment, take out the cell plate from the incubator, add 10% of the original culture volume of lactate dehydrogenase (LDH) release agent to the maximum enzyme activity control well, mix well and continue incubation.

[0057] (4) Aspirate 120 μL of the culture supernatant and use the C0017 LDH Cytotoxicity Detection Kit from Beyotime to detect the LDH release level in the cell supernatant. Add 20 μL each of lactic acid solution, LDH solution, and INT solution to a 96-well ELISA plate, incubate in the dark for 30 min, and then measure the cytotoxicity at OD = 490 nm. Among them, cytotoxicity (%) = (absorbance of treated sample - absorbance of sample control) / (absorbance of maximum enzyme activity - absorbance of sample control) × 100%.

[0058] As Figure 3 shown, after treatment with Epetraborole hydrochloride at concentrations of MIC, 5MIC, and 10MIC for 3 h, there was no significant difference in LDH cytotoxicity compared with the untreated group (Control group), and the cytotoxicity was low, indicating that the small molecule compound Epetraborole hydrochloride has good safety.

[0059] Example 5 Evaluation of the antibacterial activity of the small molecule compound Epetraborole hydrochloride at the cellular level

[0060] (1) Seed J774A.1 cells into a 48-well plate at a density of 1×10 5 cells per well, with a culture medium volume of 300 μL per well, and culture at 37 °C in 5% CO2 until the next day.

[0061] (2) Pick a single colony of Salmonella enteritidis Z11 from the LB plate and inoculate it into 1 mL of liquid LB medium. Culture it overnight at 37 °C with 180 rpm.

[0062] (3) Expand the overnight-cultured bacteria into two EP tubes, incubate them statically at 37 °C for 3 h, centrifuge at 5000 rpm for 5 min at 4 °C, discard the supernatant, and resuspend with 1 mL of PBS. Adjust the washed bacterial suspension to OD 600 = 1.0.

[0063] (4) Add 200 μL of the bacterial suspension diluted with Opti-MEM medium at a ratio of MOI = 100 to each well of J774A.1 cells. At the same time, add small molecule compounds at different concentrations (MIC, 5MIC, 10MIC), and measure the cytotoxicity according to the method of Example 4. Among them, Z11 represents J774A.1 cells after being infected with Salmonella enteritidis Z11.

[0064] As Figure 4 shown, after being infected with Salmonella enteritidis Z11, the cytotoxicity of J774A.1 cells is significantly enhanced, indicating that Salmonella enteritidis infection can enhance cytotoxicity. Compared with the group infected with Salmonella enteritidis alone, as the drug concentration increases, the cytotoxicity gradually decreases. The above results show that the small molecule drug Epetraborole hydrochloride has significant antibacterial activity on the J774A.1 cell model. It shows that the small molecule compound Epetraborole hydrochloride has significant antibacterial activity in vitro.

[0065] Example 6 Determination of the adhesion and invasion rate of the small molecule compound Epetraborole hydrochloride at the cellular level

[0066] 1. Bacterial in vitro adhesion experiment:

[0067] (1) Inoculate a single colony of Salmonella enteritidis Z11 into the corresponding LB medium and culture it overnight. Expand the overnight-cultured bacteria at a ratio of 1:100 in 3 mL of LB medium, incubate it statically at 37 °C for 3.5 h, and then adjust OD 600 = 1.0.

[0068] (2) Inoculate J774A.1 cells into a 24-well plate at a density of 2×10 5 per well and culture it overnight. After discarding the cell supernatant, wash it once with Dulbecco's phosphate buffered saline (D-PBS), and add 300 μL of Opti-MEM medium containing the secondarily cultured bacteria at a ratio of MOI = 100 (the concentration of bacteria is 1×10 9(CFU / mL), were mixed with drug concentrations of MIC, 5MIC, and 10MIC respectively. The control group was only added with the bacterial solution, and then placed in a 37°C 5% CO2 cell culture incubator for infection for 0.5 h.

[0069] (3) Discard the cell supernatant, wash twice with D-PBS, and add 500 μL of PBS containing 1% Triton X-100 to lyse the cells. Collect the lysate and dilute it to 10 -5 CFU, and take 10 μL of each for plate counting. Calculate the adhesion rate = (number of bacteria adhering to cells / initial number of bacteria) × 100%.

[0070] 2. Bacterial invasion experiment in vitro:

[0071] (1) The infection procedure was the same as the adhesion experiment. After 0.5 h of bacterial infection, discard the cell supernatant, wash once with D-PBS, add 1 mL of Opti-MEM containing 100 μg / mL gentamicin, and continue the infection for 1 h. Discard the cell supernatant, wash twice with D-PBS, and add 500 μL of PBS containing 1% Triton X-100 to lyse the cells. The dilution and plate counting steps were the same as the adhesion experiment.

[0072] (2) Calculate the invasion rate I, and the formula is I = (E / S) × 100%; E is the number of bacteria invading cells, and S is the initial number of bacteria.

[0073] As Figure 5 shown, after infection with Salmonella enteritidis Z11, the adhesion and invasion rates of J774A.1 cells were significantly enhanced, indicating that Salmonella enteritidis infection can enhance the adhesion and invasion ability to cells ( Figure 5 the left figure in Figure 5 is the statistical chart of the adhesion rate;

[0074] the right figure in

[0075] is the statistical chart of the invasion rate). Compared with the group infected with Salmonella enteritidis alone (Z11), with the increase of the drug concentration, the adhesion and invasion ability of bacteria to cells gradually decreased. The above results indicate that the small molecule drug Epetraborole hydrochloride has significant antibacterial activity on the J774A.1 cell model.

[0076] Pick a single colony of Salmonella enteritidis Z11 from the LB plate and inoculate it into 1 mL of LB medium, and culture overnight at 37°C with 180 rpm. Add the small molecule compound Epetraborole hydrochloride at a concentration of 1 / 5MIC to the overnight cultured bacteria, and expand the culture for 4 h until OD600 = 0.8. The bacterial solution was centrifuged at 5000 rpm for 5 min at 4°C, the supernatant was discarded, and the bacterial cell pellet was collected. The collected pellet was used to extract bacterial RNA using Cell / Tissue Total RNA Isolation Kit V2.

[0077] Using III RT SuperMix for qPCR(+gDNA wiper), the RNA was reverse-transcribed into cDNA. The first step was the genomic DNA removal reaction: 1 μg of RNA template, 4 μL of 4×gDNA wiper Mix was added, and RNase-free H2O was used to make up to 12 μL. The reaction conditions were 42°C for 2 min. The second step was the reverse transcription reaction. The reaction solution from the first step was added with 4 μL of 5×HiScript III qRT SuperMix, pipetted and mixed well, incubated at 37°C for 15 min, and the reaction was terminated at 85°C for 5 s. The obtained cDNA was stored at -80°C for later use.

[0078] II. qRT-PCR

[0079] The qRT-PCR reaction system (20 μL) was as follows: 10 μL of 2×Universal SYBR Master Mix, 6.8 μL of RNase-free H2O, 0.6 μL of upstream / downstream primers (Table 2, 10 μM each), 2 μL of diluted cDNA (40 ng). The reaction mixture was placed in a QuantStudio 6Flex thermal cycler for PCR amplification. The qRT-PCR reaction program was: pre-denaturation at 37°C for 30 sec; 95°C for 5 sec; 60°C for 60 sec, for 40 cycles. Using the housekeeping gene gyrB (ID: 1255362) as an internal reference, the relative expression level was calculated using the log2(2 -ΔΔCT ) method.

[0080] As Figure 6 shown, after adding a drug concentration of 1 / 5 MIC (0.096 μM), the expression levels of T3SS1-related genes (Table 3) were significantly down-regulated, indicating that the small molecule compound Epetraborole hydrochloride could target and inhibit the expression of T3SS1, playing a role in preventing and controlling Salmonella.

[0081] Table 2 shows the primer sequences required for fluorescence quantification

[0082]

[0083] Table 3 shows the accession numbers of T3SS1-related genes

[0084]

[0085] Example 8 Antibacterial Effect of Small Molecule Compound Epetraborole Hydrochloride on Salmonella of Different Sources and Different Serotypes

[0086] (1) Pick single colonies of Salmonella enteritidis from different sources and other serotypes (Table 4) and inoculate them into MH liquid medium respectively, and culture overnight at 37°C with 180 rpm;

[0087] (2) Centrifuge the bacterial liquid at 5000 rpm for 5 min at 4°C, adjust the bacterial OD 600 = 1, and then dilute the bacterial liquid to 2×10 5 CFU / mL.

[0088] (3) Take a sterile 96-well plate and perform drug dilution in a biosafety cabinet. The MIC value is in the sixth column. Dilute the drug 2-fold in the first to tenth columns in the order of decreasing concentration, add 100 μL to each well, and the eleventh column is the control group without drug.

[0089] (4) Add 100 μL of the diluted bacterial liquid to each well, and the twelfth column is the control group without bacteria. Place the 96-well plate in an incubator at 37°C for 12 hours, and observe whether the Salmonella enteritidis from different sources has antibacterial effects within the MIC concentration range. A value below 5MIC is judged to have antibacterial performance.

[0090] Table 4 shows the inhibitory effect of small molecule compound Epetraborole hydrochloride on Salmonella enteritidis of different sources and different serotypes

[0091]

[0092]

[0093]

[0094] As shown in Table 4, the small molecule compound Epetraborole hydrochloride has good antibacterial effects on Salmonella of different sources and different serotypes, indicating that Epetraborole hydrochloride has potential application value for the prevention and control of Salmonella in different scenarios.

Claims

1. A pharmaceutical composition or vaccine for treating and / or inhibiting Salmonella infection, characterized in that, It contains the small molecule drug Epetraborole hydrochloride.

2. The pharmaceutical composition or vaccine according to claim 1, wherein The Salmonella is Salmonella enteritidis, Salmonella typhimurium, Salmonella london, Salmonella derby, Salmonella kentucky or Salmonella rosen.

3. The pharmaceutical composition or vaccine according to claim 1, characterized in that, The dosage form of the pharmaceutical composition includes tablets, capsules, granules, aerosols, sprays or injections.

4. Use of the small molecule compound Epetraborole hydrochloride in the preparation of a pharmaceutical composition or vaccine for treating and / or preventing Salmonella infection.

5. The application according to claim 4, characterized in that The Salmonella is Salmonella enteritidis, Salmonella typhimurium, Salmonella london, Salmonella derby, Salmonella kentucky or Salmonella rosen.

6. The application according to claim 4, characterized in that The concentration of the small molecule compound Epetraborole hydrochloride is 0.24 - 0.96 μM.

7. The application according to claim 4, characterized in that, When the Salmonella is Salmonella enteritidis, the concentration of the small molecule compound Epetraborole hydrochloride is 0.24 - 4.8 μM.

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