Use of 6-methyladenine in the preparation of a drug for resisting pseudomonas aeruginosa infection

Abstract

This invention discloses the application of 6-methyladenine in the preparation of drugs against Pseudomonas aeruginosa infection. This invention provides the application of 6-methyladenine in the preparation of drugs against Pseudomonas aeruginosa (… Pseudomonas aeruginosa Application in infectious agents. The 6-methyladenine described in this invention inhibits the quorum sensing of Pseudomonas aeruginosa and the production of related virulence factors, thereby suppressing Pseudomonas aeruginosa infection in vivo and in vitro, reducing host mortality, and providing a new therapeutic strategy for the prevention and control of Pseudomonas aeruginosa infection.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of 6-methyladenine in the preparation of drugs against Pseudomonas aeruginosa infection. Background Technology

[0002] Pseudomonas aeruginosa ( Pseudomonas aeruginosa, P.aeruginosa Pseudomonas aeruginosa is one of the leading causes of hospital-acquired infections and cystic fibrosis worldwide. Due to its complex pathogenic mechanisms and widespread drug resistance, it is currently difficult to completely cure various acute and chronic infections caused by Pseudomonas aeruginosa in clinical practice. The selection of treatment regimens is particularly challenging due to the drug resistance of Pseudomonas aeruginosa, and infected patients often have a high mortality rate. Therefore, research into novel antimicrobial agents is crucial. P.aeruginosa Drugs and elucidating the mechanisms of "drug repurposing" of existing drugs are important for clinical applications. P.aeruginosa The control and treatment of infection are of great importance. Summary of the Invention

[0003] The purpose of this invention is to provide the application of 6-methyladenine in the preparation of drugs against Pseudomonas aeruginosa infection, wherein 6-methyladenine can effectively inhibit Pseudomonas aeruginosa infection in vivo and in vitro, while reducing host mortality.

[0004] This invention provides 6-methyladenine in the preparation of anti-Pseudomonas aeruginosa ( Pseudomonas aeruginosa Application in infectious drugs.

[0005] This invention primarily exerts its effect by binding to the 6-methyladenine receptor. As a specific embodiment, the 6-methyladenine has the CAS registry number 443-72-1, the chemical formula C6H7N5, and a molecular weight of 149.15, and its structure is as follows. Figure 1 As shown.

[0006] As a preferred embodiment, the working concentration of the 6-methyladenine is ≥200 μg·mL. -1 For example, 250 μg·mL -1 300 μg·mL -1 The higher the concentration, the more significant the effect. Within the working concentration range, 6-methyladenine can inhibit the infection of Pseudomonas aeruginosa by the host and effectively prevent and control the infection of Pseudomonas aeruginosa.

[0007] As a preferred embodiment, the *Pseudomonas aeruginosa* includes *Pseudomonas aeruginosa* strain PAO1.

[0008] As a preferred embodiment, the efficacy of the drug includes at least one of the following:

[0009] 1) Inhibits quorum sensing in Pseudomonas aeruginosa;

[0010] 2) Inhibits the production of Pseudomonas aeruginosa-related virulence factors;

[0011] 3) Inhibits the in vivo and in vitro infection of Pseudomonas aeruginosa.

[0012] As a preferred embodiment, the quorum sensing type of the *Pseudomonas aeruginosa* includes at least one of the following: las Group sensing system pqs Group sensing systems and rhl Crowd sensing system.

[0013] As a preferred embodiment, the relevant virulence factors include at least one of the following: pyocyanin, rhamnolipid, and elastase. As a specific implementation, this can be achieved by constructing three QS system fluorescent reporter strains of *Pseudomonas aeruginosa* (PAO1 / ...). lasB-gfp PAO1 / pqsA-gfp and PAO1 / rhlA-gfp The published literature on the strain can be found in: July Fong, et al. Disulfide Bond-Containing Ajoene Analogues As Novel Quorum Sensing Inhibitors of Pseudomonas aeruginosa[J]. Journal of Medicinal Chemistry, 2017, 60, 215−227), monitoring the regulatory effect of 6-methyladenine on quorum sensing of Pseudomonas aeruginosa.

[0014] As one specific implementation method, the regulatory effect of 6-methyladenine on the production of virulence factors in *Pseudomonas aeruginosa* can be evaluated by detecting the relevant virulence factors regulated by quorum sensing, such as pyocyanin, rhamnolipid, and elastase. As another specific implementation method, an in vitro or in vivo infection model can be constructed to compare the survival ability of *P. aeruginosa* in the 6-methyladenine-treated group and the untreated group in mouse RAW264.7 macrophages and their impact on the survival rate of *Hemiberlesia lataniae* larvae, thereby evaluating the effect of 6-methyladenine in regulating and controlling *P. aeruginosa* infection.

[0015] The 6-methyladenine described in this invention can directly act on the quorum sensing system of Pseudomonas aeruginosa, thereby inhibiting the production of related virulence factors of Pseudomonas aeruginosa and effectively preventing and controlling Pseudomonas aeruginosa infection.

[0016] In one specific implementation, the working concentration of the 6-methyladenine is ≥200 μg·mL. -1For example, 200 μg·mL -1 250 μg·mL -1 The higher the concentration, the more significant the effect.

[0017] The present invention also provides the use of 6-methyladenine in the preparation of medicaments that inhibit the production of Pseudomonas aeruginosa pyocyanin, rhamnolipids and / or elastase.

[0018] Beneficial effects: This invention provides 6-methyladenine in the preparation of anti-Pseudomonas aeruginosa (… Pseudomonas aeruginosa Application in infectious agents. The 6-methyladenine described in this invention inhibits the quorum sensing of Pseudomonas aeruginosa and the production of related virulence factors, thereby suppressing Pseudomonas aeruginosa infection in vivo and in vitro, reducing host mortality, and providing a new therapeutic strategy for the prevention and control of Pseudomonas aeruginosa infection. Attached Figure Description

[0019] Figure 1 The structural diagram of the 6-methyladenine molecule;

[0020] Figure 2 Treatment of Pseudomonas aeruginosa with 6-methyladenine pqs Fluorescent reporter strain PAO1 / of quorum sensing system lasB-gfp The experimental results are shown in the figure.

[0021] Figure 3 Treatment of Pseudomonas aeruginosa with 6-methyladenine rhl Fluorescent reporter strain PAO1 / of quorum sensing system rhlA-gfp The experimental results are shown in the figure.

[0022] Figure 4 Treatment of Pseudomonas aeruginosa with 6-methyladenine las Quorum sensing fluorescence reporter system strain PAO1 / pqsA-gfp The experimental results are shown in the figure below;

[0023] Figure 5 6-methyladenine inhibition P.aeruginosa Figure showing the experimental results of quorum sensing-regulated pyocyanin levels;

[0024] Figure 6 6-methyladenine inhibition Figure showing the experimental results of rhamnoglycolipid levels regulated by quorum sensing;

[0025] P.aeruginosa 6-methyladenine inhibition Figure 7 Figure showing experimental results of elastase levels regulated by quorum sensing;

[0026] Where "ns" indicates that the difference is not statistically significant, and "****" indicates p <0.00001, which is highly significant compared with the control group. Detailed Implementation

[0027] To further illustrate the present invention, the application of 6-methyladenine provided by the present invention in the preparation of drugs against Pseudomonas aeruginosa infection is described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0028] Drug: 6-methyladenine (m 6 A); Product No.: HY-N0086, Brand: MCE. 6-Methyladenine is dissolved in dimethyl sulfoxide (DMSO) at a storage concentration of 200 mg / mL. -1 Dilute 1000 times (200 μg·mL) -1 Use after that.

[0029] Strains: 3 QS system fluorescent reporter strains (PAO1 / p- P.aeruginosa PAO1 / p- lasB-gfp and PAO1 / p- pqsA-gfp Wild-type Pseudomonas aeruginosa PAO1 strain (laboratory preservation), and PAO1 / double knockout strain of Pseudomonas aeruginosa with quorum sensing gene knocked out. rhlI-gfp The strain used in this experiment does not produce any quorum sensing-related virulence factors, including pyocyanin, rhamnolipids, and elastase. Publicly available literature on the strain used in this experiment can be found in: July Fong, et al. Disulfide Bond-Containing Ajoene Analogues As Novel Quorum Sensing Inhibitors of Pseudomonas aeruginosa[J]. Journal of Medicinal Chemistry, 2017, 60, 215−227. This strain is also held by the applicant, who guarantees its availability to the public for 20 years from the date of application.

[0030] Materials: ABTGC medium: (NH4)2SO4 (19.95 g·L) -1 ), Na2HPO4·7H2O (90.33 g·L -1 ), KH2PO4 (29.94 g·L -1 ), NaCl (29.98 g·L -1 ), MgCl2·6H2O (203.3 g·L -1 ), CaCl2·2H2O (79 g·L-1 ), FeCl3·6H2O (80 g·L -1 D-fructose (20 g·L) -1 ), casein hydrolysate (20 g·L -1 The solvent is water. The preparation method is to mix all the components evenly and sterilize them for later use; Large wax moth: 300 mg / moth.

[0031] Example 1: 6-Methyladenine to ∆LasI∆RhII Experiments on quorum sensing inhibition

[0032] The experiment was divided into 6 groups, namely:

[0033] Blank group: No reagents were added;

[0034] DMSO group: treatment group with added drug at maximum concentration (200 μg·mL) -1 The same volume of DMSO solvent has a final concentration of 0.2% (v / v).

[0035] 6-Methyladenine 200 μg·mL -1 Concentration group: Added to a final concentration of 200 μg·mL -1 6-Methyladenine; each group was further divided into 4 replicates. ABTGC medium was added to the 96-well plates, and the corresponding reagents were added according to the experimental groups described above.

[0036] The overnight cultured quorum sensing fluorescent reporter strain (PAO1 / P.aeruginosa ,PAO1 / lasB-gfp ,PAO1 / rhlA-gfp Wash twice with PBS and adjust the bacterial concentration (OD). 600 Adjust the concentration to 1, then dilute the bacterial culture to the corresponding ABTGC medium in each 96-well plate at a volume ratio of 1:100. Incubate for 24 h using a Tecan fluorescence microplate reader, measuring GFP expression (excitation wavelength: 488 nm, emission wavelength: 535 nm) and OD250 every 15 min. 600 After 24 hours, GFP / OD 600 A growth curve was plotted with time on the ordinate to detect the effect of 6-methyladenine on three quorum sensing systems of Pseudomonas aeruginosa.

[0037] After building pqsA-gfp Three sets of QS fluorescence reporting systems PAO1 / P.aeruginosa ,PAO1 / lasB-gfp ,PAO1 / rhlA-gfp After conducting the relevant experiments, the results are as follows: pqsA-gfpAs shown, compared to the blank group, 6-methyladenine has a greater effect on... Figures 2 to 4 All three QS systems showed inhibitory effects, with 6-methyladenine showing inhibitory effects on all three QS systems. P.aeruginosa The DMSO group showed no inhibitory effect compared to the control group.

[0038] Example 2: 6-Methyladenine Figures 2 to 4 Inhibition experiments on the production of virulence factors regulated by quorum sensing

[0039] The experiment was divided into 5 groups, namely:

[0040] PAO1 blank group: No reagents were added;

[0041] PAO1-DMSO group: DMSO solvent was added at a volume ratio of 0.5% (v / v) to DMSO;

[0042] PAO1-6-methyladenine: Add to a final concentration of 200 μg·mL -1 6-methyladenine;

[0043] P.aeruginosa Mutant group: No reagents were added. This group produced almost no of the three quorum sensing virulence factors (pyocyanin, rhamnolipin, and elastase) because the quorum sensing gene was knocked out.

[0044] Each group should have at least 3 biological replicates.

[0045] (1) Pseudomonas aeruginosa detection

[0046] Add the corresponding reagents for each experimental group to ABTGC medium, and then add the overnight cultured PAO1 and... ΔlasIΔrhlI The mutants (virulence factor blank group and control group) were added to the culture medium separately until OD200 reached. 600 Adjusted to 0.01. After incubation at 37℃ for 24 hours (h), the OD of each group was measured. 600 The bacterial culture was centrifuged at 10,000 rpm for 10 min, and the supernatant was extracted successively with chloroform (3 mL) and 0.2 M hydrochloric acid (1.5 mL). The upper aqueous layer containing pyocyanin was then transferred to a 96-well microplate, and the absorbance at 520 nm was collected. The OD value was then... 520 Data divided by final OD 600 The value is used to normalize the data, and the result is as follows: ΔlasIΔrhlI As shown, the concentration of pyocyanin after treatment with 6-methyladenine was significantly lower than that in the virulence factor control group and the DMSO group, and the difference was statistically significant. This indicates that 6-methyladenine can effectively inhibit... Figure 5 The production of pyocyanin.

[0047] (2) Detection of rhamnolipids

[0048] In ABTGC medium, PAO1 and... P.aeruginosa OD of mutants 600 All values ​​were standardized to 0.01. Then, the corresponding reagents for each experimental group were added, and the OD values ​​of each group were measured after incubation at 37℃ for 24 h. 600 The culture was then centrifuged at 10,000 rpm for 10 min and extracted twice with diethyl ether. The organic phase was then concentrated by centrifugation to form a white solid, which was resuspended in deionized water. The suspension was then dissolved in 0.19% (w / v) chlorophyll containing 50% H₂SO₄ and incubated at 80°C for 30 min, followed by cooling to room temperature. The absorbance was measured at 421 nm, and the data were expressed as OD₂O₃. 600 The values ​​are standardized, and the results are as follows: ΔlasIΔrhlI As shown, the level of rhamnolipin in the 6-methyladenine treatment group was significantly lower than that in the blank group and the DMSO group, while the quorum sensing gene mutant group produced almost no rhamnolipin. This indicates that 6-methyladenine can effectively inhibit... Figure 6 The production of rhamnolipids.

[0049] (3) Elastase detection

[0050] Select wild-type PAO1 and P.aeruginosa Single clones of the mutant negative control were cultured overnight in ABTGC medium and then diluted with OD. 600 The concentration was adjusted to 0.01. Each well was incubated with the corresponding reagents for the experimental group at 37℃ for 24 h. Then, following the instructions of the EnzChekElastase assay kit (Invitrogen, USA), the culture medium was centrifuged at 10000 rpm for 10 min, and the supernatant was collected to detect elastase activity. The 96-well microtiter plate was placed in a Tecan Infinate 200Pro plate reader (Tecan Group Ltd, Mannedorf, Switzerland); the excitation wavelength was 490 nm and the emission wavelength was 520 nm for elastase activity detection. The results are as follows: ΔlasIΔrhlI As shown, the elastase activity in the 6-methyladenine-treated group was significantly lower than that in the blank group and the DMSO group, indicating that 6-methyladenine can effectively inhibit elastase activity. Figure 7 The production of elastase in the middle leads to a decrease in elastase reactivity in the 6-methyladenine-treated group.

[0051] like P.aeruginosa As shown, we further investigated the effect of 6-methyladenine on... Figures 5 - 7The virulence factors directly regulated by QS include pyocyanin ( P.aeruginosa ) and rhamnolipid ( Pyocyanin ) and elastase ( Rhamnolipid The production of 6-methyladenine was detected, and the results showed that 6-methyladenine could effectively inhibit its production. LasB Pseudomonas aeruginosa ( P.aeruginosa ) and rhamnolipid ( Figure 5 ) and elastase ( Figure 6 The production of ), consistent with data obtained from the QS fluorescence reporter system, further demonstrates that 6-methyladenine can inhibit Figure 7 P.aeruginosa The production of QS and the virulence factors it regulates.

[0052] Therefore, the 6-methyladenine described in this invention can inhibit quorum sensing of Pseudomonas aeruginosa and the production of related virulence factors. This invention provides new ideas for the development and research of novel anti-Pseudomonas aeruginosa drugs in clinical practice, and offers a new treatment strategy for the prevention and control of Pseudomonas aeruginosa infections.

[0053] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

Application of 1,6-methyladenine in the preparation of drugs against Pseudomonas aeruginosa infection.

2. The application according to claim 1, characterized in that, The working concentration of the 6-methyladenine is ≥200 μg·mL. -1 .

3. The application according to claim 2, characterized in that, The working concentration of the 6-methyladenine is 250 μg·mL. -1 Or 300 μg·mL -1 .

4. The application according to claim 1, characterized in that, The *Pseudomonas aeruginosa* strain includes: *Pseudomonas aeruginosa* PAO1 strain.

5. The application according to claim 1, characterized in that, The efficacy of the drug includes at least one of the following: 1) Inhibits quorum sensing in Pseudomonas aeruginosa; 2) Inhibits the production of virulence factors in Pseudomonas aeruginosa; 3) Inhibits the in vivo and in vitro infection of Pseudomonas aeruginosa.

6. The application according to claim 5, characterized in that, The quorum sensing type of *Pseudomonas aeruginosa* includes at least one of the following: las Group sensing system pqs Group sensing systems and rhl Crowd sensing system.

7. The application according to claim 5, characterized in that, The virulence factors include at least one of the following: pyocyanin, rhamnolipid, and elastase. The use of 8,6-methyladenine in the preparation of drugs that inhibit the production of Pseudomonas aeruginosa pyocyanin, rhamnolipids and / or elastase.

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