Application of two small molecule compounds in prevention and treatment of brucellosis

Abstract

The invention discloses an application of AMG (Amplified Macrocaryon Gamma) 837calcia hydrogen and ROR [gamma] t invase agonist 13 in the aspect of preparing a medicine for preventing or treating brucellosis, which is characterized in that the AMG 837calcia hydrogen and the ROR [gamma] t invase agonist 13 are used as raw materials; according to the present invention, the drug sensitive paper diffusion method in vitro inhibition experiment results show that the compounds AMG 837calcia hydrochloride and ROR [gamma] t invase agonist 13 have good in vitro inhibition effects on Brucella, and the cytotoxicity detection results show that the AMG 837calcia hydrochloride and ROR [gamma] t invase agonist 13 have no toxicity on the Brucella host cell RAW264.7 under the prevention or treatment dose; the AMG 837calcia hydrogen and the ROR [gamma] t invaded agonist 13 can be used for effectively eliminating intracellular parasitic bacterium brucella infected cells and intracellular colonization at a cellular level. Therefore, the AMG 837calcia hydrate and the ROR [gamma] t invase agonist 13 can be used for preparing the medicine for resisting the brucella infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of the use of compound drugs, and particularly relates to the application of two small molecule compounds, AMG837 calcium hydrate and RORγt inverse agonist 13, in the preparation of drugs for preventing or treating brucellosis. Background Art

[0002] Brucellosis is a serious burden on the global economy and public health security. One of the most important characteristics of Brucella is that they are facultative intracellular parasites, mainly invading the host's macrophages and trophoblast cells, and affecting host cell damage and apoptosis through specific molecular mechanisms, mediating autophagy of host cells, thus facilitating their survival and reproduction in host cells. Although there are already a variety of drugs available for the treatment of Brucella infections, in recent years, the drug resistance of the vast majority of bacteria has shown an upward trend, and low antibiotic cure rates and infection relapses have also been frequently reported in humans and livestock. In summary, there is an urgent need in this field to develop new antibacterial drugs.

[0003] AMG 837 calcium hydrate is an effective and orally active partial agonist of GPR40 / FFA1, inhibiting 3 the specific binding of [[H]]AMG 837 to the human FFA1 receptor, with a pIC 50 value of 8.13. It can also enhance insulin secretion in rodents and reduce glucose levels. RORγt inverse agonist 13 is an effective, orally active, and selective RORγt inverse agonist, with an IC 50 value of 63.8 nM. There is currently no report on the antibacterial activity of AMG 837 calcium hydrate and RORγt inverse agonist 13, and there is no clinical development and application information. Summary of the Invention

[0004] The purpose of the present invention is to provide the use of two small molecule compounds, AMG 837 calcium hydrate and RORγt inverse agonist 13, in the preparation of drugs for preventing and treating Brucella infections.

[0005] To solve the above problems, the present invention provides the following technical solutions:

[0006] The application of AMG 837 calcium hydrate in the preparation of antibacterial drugs, characterized in that the chemical structure of the AMG 837 calcium hydrate is as follows:

[0007]

[0008] Use of RORγt inverse agonist 13 in the preparation of an antibacterial infection drug, characterized in that the chemical structure of the RORγt inverse agonist 13 is as follows:

[0009]

[0010] Furthermore, the bacterium is Brucella.

[0011] Compared with the prior art, the present invention has the following beneficial effects: AMG 837 calcium hydrate and RORγt inverse agonist 13 have antibacterial activity and can be used in the preparation of drugs related to the prevention or treatment of brucellosis, providing new uses for AMG 837 calcium hydrate and RORγt inverse agonist 13. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Structural formula of AMG 837 calcium hydrate.

[0013] Figure 2 Structural formula of RORγt inverse agonist 13.

[0014] Figure 3 Detection results of the maximum inhibitory diameter of AMG 837 calcium hydrate against various species of Brucella in Example 1.

[0015] Figure 4 Detection results of the maximum inhibitory diameter of RORγt inverse agonist 13 against various species of Brucella in Example 1.

[0016] Figure 5 Detection results of the drug resistance phenotype of AMG 837 calcium hydrate against various species of Brucella in Example 2.

[0017] Figure 6 Detection results of the drug resistance phenotype of RORγt inverse agonist 13 against various species of Brucella in Example 2.

[0018] Figure 7 Detection results of the safety of AMG 837 calcium hydrate on RAW264.7 cells in Example 3.

[0019] Figure 8Detection results of the safety of RORγt inverse agonist 13 on RAW264.7 cells in Example 3.

[0020] Figure 9 shows the detection results of the ability of AMG 837 calcium hydrate and RORγt inverse agonist 13 to inhibit the intracellular invasion of Brucella in Example 4. Figure 9A Colony counting results of AMG 837 calcium hydrate Figure 9B Colony counting results of RORγt inverse agonist 13 Figure 9C Orthogonal fluorescence microscopy examination results of the inhibition of intracellular Brucella by AMG 837 calcium hydrate and RORγt inverse agonist 13 after 24 hours of infection. Detailed implementation manners

[0021] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. The implementation manners of the present invention include but are not limited to the following examples.

[0022] Among them, AMG 837 calcium hydrate and RORγt inverse agonist 13 can inhibit the activity of Brucella. The following are the activity experiments of AMG 837 calcium hydrate and RORγt inverse agonist 13 against Brucella.

[0023] Example 1 Maximum inhibitory diameter of AMG 837 calcium hydrate and RORγt inverse agonist 13 against various species of Brucella

[0024] Experimental materials:

[0025] Reagents: AMG 837 calcium hydrate and RORγt inverse agonist 13 (purchased from Shanghai Aoyuansheng Biotechnology Co., Ltd.). CAS numbers are 1259389-38-2 and 2170477-75-3 respectively. The control drugs for all experiments are rifampicin (RIF) or gentamicin (GM) (purchased from Shanghai Aoyuansheng Biotechnology Co., Ltd.).

[0026] Strains: Brucella melitensis TZ, Brucella melitensis M5, Brucella suis S2, and Brucella abortus A19.

[0027] Experimental method:

[0028] Taking Brucella melitensis TZ as an example, the Brucella melitensis TZ bacterial solution was cultured and diluted to 1×10 6 CFU / mL. 100 μL of the bacterial solution was inoculated onto an LB solid medium by the spread plate method. The AMG 837 calcium hydrate and RORγt inverse agonist 13 solutions were respectively diluted to 5 mg / mL. 10 μL was dropped onto each sterile filter paper and pasted onto the TSA plate inoculated with Brucella melitensis TZ. 3 identical drug sensitivity test papers were pasted on each TSA plate for parallel experiments. The distance between every two drug sensitivity test papers was not less than 24 mm, and the center of the drug sensitivity test paper was not less than 15 mm from the edge of the plate. After incubation in an incubator at 37°C and 5% CO 2 for 72 h, the maximum inhibitory diameter of each drug sensitivity test paper was accurately measured with a vernier caliper.

[0029] The test results are shown in Figure 3 and Figure 4 , and it can be found that AMG 837 calcium hydrate and RORγt inverse agonist 13 can significantly inhibit the growth of Brucella of various species.

[0030] Example 2 Minimum inhibitory concentration of AMG 837 calcium hydrate and RORγt inverse agonist 13 against Brucella of various species

[0031] Taking Brucella melitensis TZ as an example, a 96-well bacterial culture plate was taken, 1.5×10 8The Brucella melitensis TZ dilution solution at CFU / mL is ready for use. Under aseptic conditions, add 100 μL of TSB liquid medium to each well in rows 1 - 4. Add 100 μL of AMG 837 calcium hydrate and RORγt inverse agonist 13 at a concentration of 256 μg / mL to the first 4 wells in the first column respectively, and pipette up and down evenly. Take 100 μL of liquid from each well in the first column and vertically and suspendedly pipette it parallelly to the second column and pipette up and down evenly. Repeat the above operation steps until it is pipetted to the tenth column and pipetted up and down evenly. Then, draw 100 μL of the mixed solution from each well in the tenth column and discard it. Add 200 μL of TSB liquid medium to the twelfth column as a blank control. Add 100 μL of the Brucella melitensis TZ dilution solution to the first 3 rows in columns 1 - 11 respectively and pipette up and down evenly. Among them, the eleventh column is a blank control of 100 μL of bacterial solution + 100 μL of TSB liquid medium, and the fourth row is a blank control of TSB liquid medium + AMG837 calcium hydrate or RORγt inverse agonist 13. Finally, place the culture plate in a 37°C, CO 2 After 24 h in an incubator, observe the results.

[0032] The test results are shown in Figure 5 and Figure 6 It can be found that each strain is sensitive to AMG 837 calcium hydrate and RORγt inverse agonist 13.

[0033] Example 3 Safety effects of AMG 837 calcium hydrate and RORγt inverse agonist 13 on Brucella host cells

[0034] Mouse macrophage RAW264.7 is stored in liquid nitrogen, taken out and resuscitated, and passaged continuously for three generations. After the cells grow well, they are used for experimental research. Inoculate RAW264.7 cells into a 96-well plate, with the cell amount in each well being 1×10 5 , at 37°C, 5% CO 2Cultivate overnight in a constant temperature cell incubator. When the cell density reaches 70 - 90%, add 100 μL / well of 10% FBS DMEM containing different concentrations of AMG 837 calcium hydrate and RORγt inverse agonist 13 (starting concentration 1024 μg / mL, serially dilute the test drug with a 2-fold gradient). Set up 5 replicates for each concentration. Additionally, set up blank controls and PBS controls with 4 replicates, continue the culture, and observe the cell status under a microscope at different time intervals. After adding the drug for 24 h, add 10 μL of CCK solution to each well and incubate at 37 °C and 5% CO 2 for 1 h under the given conditions, and measure the OD value at 450 nm. Analyze the data using Graphpad Prism 8.0 software and calculate the half-maximal inhibitory concentration (IC 50 ) of the drug on the cells.

[0035] The detection results are shown in Figure 7 and Figure 8 . After analysis, it was found that AMG 837 calcium hydrate and RORγt inverse agonist 13 had no effect on RAW264.7 cells at treatment concentrations below or equal to 16 μg / mL.

[0036] Example 4 Inhibitory effect of AMG 837 calcium hydrate and RORγt inverse agonist 13 on the intracellular invasion ability of Brucella

[0037] Taking Brucella melitensis TZ labeled with green fluorescence as an example, take RAW264.7 mouse macrophages frozen in liquid nitrogen, quickly shake them in a 37 °C water bath until completely melted, centrifuge at 500×g for 3 min, aspirate the supernatant, resuspend with 10% FBS-DMEM (antibiotic-free) medium, transfer to a T25 culture flask, gently pipette to mix evenly, and place in a CO 2 incubator at 37 °C for culture. After the cells are passaged continuously 2 - 3 times, inoculate them into a 24-well cell culture plate at a density of 1×10 5 / well, allow them to adhere and differentiate into macrophages. Take 1 mL of Brucella broth at the exponential phase with OD 600 = 0.6 - 0.8, centrifuge at 8000×g for 3 min, discard the supernatant, resuspend the bacteria with 1 mL of DMEM medium, centrifuge at 8000×g for 3 min, and repeat this operation three times. Resuspend the bacteria with 2% FBS-DMEM medium and dilute to an appropriate concentration for standby. CO 2Take out the 24-well plate from the incubator, aspirate and discard the supernatant, and wash twice with PBS. Infect RAW264.7 cells with the pre-diluted bacterial solution at an MOI of 100:1, centrifuge at 400×g for 5 min, and record the infection time as 0 h at this time; place the cell plate at 37 °C and 5% CO 2 Incubate under the conditions for 1 h to allow the bacteria to fully adsorb and infect RAW264.7 cells. After 1 h, rinse each well 2 times with PBS to wash away the extracellular bacteria of uninfected cells, and be careful to operate gently to prevent blowing up the cells. Add 1 mL of 10% FBS-DMEM medium containing 10 μg / mL gentamicin to each well and place it at 37 °C and 5% CO 2 Act under the conditions for 1 h, aspirate and discard the culture medium, wash the cells 2 times with PBS, and finally add maintenance medium containing different concentrations of AMG 837 calcium hydrate and RORγt inverse agonist 13 (starting from a concentration of 1 μg / mL, increasing by 2-fold concentration respectively, with the highest concentration of 16 μg / mL) and continue to incubate at 37 °C and 5% CO 2 Incubate under the conditions. The colony counting experiment sets up a blank control group without using AMG 837 calcium hydrate and RORγt inverse agonist 13, and samples are collected at 0 h, 6 h, 12 h, 24 h, and 48 h respectively for CFU counting. The operation is as follows: rinse each well 2 times with PBS, add 1 mL of 0.2% Triton X-100 solution to fully lyse the cells for 10 min, dilute to an appropriate concentration in gradient, and then take 100 μL of the diluted solution and evenly coat it on the TSA plate without antibiotics. After the single colonies grow, calculate the original CFU in each well according to the number of colonies and the dilution factor, and analyze the intracellular proliferation ability of the isolated strain. For the upright fluorescence microscopy examination experiment, a microscopy coverslip needs to be placed in each well of the cell culture plate in advance. After infecting with fluorescent bacteria for 24 hours, rinse each well 2 times with PBS, add 1 mL of electron microscope fixing solution and fix it at room temperature in the dark for 30 min, then clip out the coverslip and place it on a glass slide dropped with anti-fluorescence quenching agent, make marks and use it for upright fluorescence microscopy examination and observation. All cell infection experiments are independently repeated 3 times.

[0038] The statistical results are shown in Figure 9. It can be found that AMG 837 calcium hydrate and RORγt inverse agonist 13 at a concentration of 16 μg / mL have an intracellular killing effect on Brucella at the cellular level, and can completely clear Brucella-infected cells and intracellular colonization.

[0039] 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 foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features, but these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of AMG 837calcium hydrate in the preparation of a drug for resisting bacterial infection, characterized in that: The chemical structural formula of the AMG 837calcium hydrate is as follows:

2. Use of RORγt inverse agonist 13 in the preparation of a drug for anti-bacterial infection, characterized in that the chemical structure of the RORγt inverse agonist 13 is as follows:

3. The use according to claims 1 and 2, characterized in that: The bacterium is Brucella.

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