Use of quercetin in the preparation of tet-x4 enzyme inhibitors

Abstract

The present application relates to the application of quercetin in the preparation of Tet-X4 enzyme inhibitors, and belongs to the field of medicine and pharmacy. The minimum inhibitory concentration test, time-kill curve detection, cytotoxicity test and treatment effect on mouse systemic infection prove that quercetin combined with tetracycline antibiotics (tigecycline, minocycline, metacycline and tetracycline) has synergistic therapeutic effect on Tet-X4 pathogenic bacteria infection in vivo and in vitro. The present application inhibits the high water resistance of Tet-X4 mediated bacteria to tetracycline antibiotics by adding quercetin, thereby restoring the antibacterial effect of tetracycline antibiotics. Quercetin does not have synergistic antibacterial effect with other types of antibiotics (such as cephalosporins, gentamicin and polymyxin). This finding has very important clinical application value, and will provide active ingredients and test basis for developing quercetin as a tetracycline antibiotic synergist.

Description

Technical Field

[0001] This invention belongs to the field of medical pharmaceuticals, specifically relating to the application of quercetin in the preparation of Tet-X4 enzyme inhibitors. Background Technology

[0002] Quercetin is a natural flavonol compound with the molecular formula C. 15 H 10 O7, possessing a typical flavonoid skeleton structure, with multiple phenolic hydroxyl groups contributing its strong biological activity, is a yellow powder or needle-like crystal with a melting point of 314℃. It is almost insoluble in water but soluble in organic solvents such as ethanol and methanol. Its alkaline aqueous solution is yellow, and it exhibits blue fluorescence under ultraviolet light. Quercetin is widely found in plants, with high content in fruits and vegetables such as onions, apples, tea, and hawthorn. It is also abundant in about 100 medicinal plants, including Sophora japonica buds and ginkgo leaves. Small amounts can be obtained through consuming these foods in daily life. Pharmacological studies have shown that quercetin has extremely strong antioxidant capacity, 50 times that of vitamin E and 20 times that of vitamin C, effectively scavenging free radicals. It also has anti-inflammatory and anti-tumor effects, inhibiting platelet aggregation and lowering blood lipids. It has adjunctive therapeutic effects on chronic bronchitis, coronary heart disease, and hypertension. Recent studies have found that its combination with dasatinib can eliminate senescent cells, providing a new direction for anti-aging research. However, its potential as a Tet-X4 enzyme inhibitor has not been reported.

[0003] Tetracycline antibiotics are a class of broad-spectrum antibiotics with a tetraphenyl shell skeleton. The first generation consists of natural products, including chlortetracycline, oxytetracycline, and tetracycline. These are widely used due to their broad antibacterial spectrum and low cost, but they are prone to developing resistance and have numerous side effects. The second generation consists of semi-synthetic derivatives, represented by doxycycline and minocycline. These are more lipophilic, more easily absorbed by cells, and have significantly improved stability and pharmacokinetic properties. The third generation, represented by tigecycline, can overcome drug-resistant bacteria and has a broader antibacterial spectrum and stronger activity. Currently, they are used as a "last line of defense" in clinical practice to treat infections caused by complex drug-resistant Gram-negative bacteria.

[0004] Tetracycline antibiotics primarily exert their bactericidal effect by binding to the A site of the 30S ribosomal subunit in bacteria, preventing the binding of aminoacyl-tRNA, and inhibiting bacterial protein synthesis. At high concentrations, they can also be bactericidal. Their antibacterial spectrum covers Gram-positive and Gram-negative bacteria, as well as rickettsiae, mycoplasma, chlamydia, and spirochetes. Bacterial resistance mechanisms to tetracycline antibiotics mainly include: active efflux systems, ribosomal protective proteins, and the production of resistance enzymes. Active efflux mechanisms are mediated by Tet-series efflux pumps encoded by plasmids or transposons. These pumps actively expel intracellular drugs, reducing drug concentration and evading their effect. Ribosomal protective proteins, by binding to the 30S ribosomal subunit, prevent tetracycline from binding to its target site, interfering with the drug's inhibitory effect on protein synthesis. It is noteworthy that bacterial resistance mediated by both active efflux and ribosomal protective protein mutations is at a low level. Bacteria can produce inactivating enzymes that chemically modify drugs, such as the Tet-X4 resistance enzyme discovered in Gram-negative pathogens in recent years. Tet-X4 resistance enzymes can mediate high levels of bacterial resistance to novel tetracyclines, including tigecycline and omalicycline. Therefore, screening for Tet-X4 inhibitors is one of the feasible strategies to address the Tet-X4-mediated tetracycline antibiotic resistance crisis. Summary of the Invention

[0005] The quercetin molecule described in this invention has the following molecular formula: C0 15 H 10 O7 has a molecular weight of 302.24.

[0006]

[0007] This invention discovers through screening that quercetin can significantly inhibit the biological activity of Tet-X4 resistant enzymes, thus providing the application of quercetin in the preparation of Tet-X4 enzyme inhibitors.

[0008] Specifically, the quercetin can significantly inhibit the activity of Tet-X4 enzyme in hydrolyzing tetracycline antibiotics, thereby restoring the antibacterial activity of tetracycline antibiotics against Tet-X4 positive pathogens.

[0009] The Tet-X4 positive pathogens are Tet-X4 enzyme-producing Escherichia coli, Klebsiella pneumoniae, and Salmonella; the tetracycline antibiotics are tigecycline, minocycline, methacycline, and tetracycline. Quercetin can enhance the antibacterial activity of tigecycline, minocycline, methacycline, or tetracycline against Tet-X4 positive pathogens; however, it does not have any synergistic antibacterial effect with other antibiotics such as ceftriaxone, gentamicin, and polymyxins.

[0010] Furthermore, the present invention is specifically applied in the preparation of a drug for treating diseases caused by Tet-X4 positive pathogens by combining quercetin with tetracycline antibiotics.

[0011] The positive effects of this invention are as follows:

[0012] This invention reveals that quercetin can inhibit the activity of the Tet-X4 enzyme, restoring the bactericidal activity of tetracycline antibiotics (tigecycline, minocycline, methacycline, and tetracycline) against Tet-X4-producing pathogens. In in vivo experiments, quercetin combined with tetracycline antibiotics showed good therapeutic effects against Tet-X4-expressing bacterial infections, demonstrating broad medical applications. This provides a safer and more reliable combination drug formulation for the treatment of complex drug-resistant bacterial skin or soft tissue infections with tetracycline antibiotics, especially tigecycline. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0014] Figure 1 Detection of quercetin's inhibition of Tet-X4 enzyme activity.

[0015] Figure 2 Time-bactericidal curves of quercetin combined with tigecycline against Tet-X4 positive bacteria.

[0016] Figure 3 Time-bactericidal curves of quercetin combined with minocycline against Tet-X4 positive bacteria.

[0017] Figure 4 Time-bactericidal curve of quercetin combined with metacycline against Tet-X4 positive bacteria.

[0018] Figure 5 Time-bactericidal curve of quercetin combined with tetracycline against Tet-X4 positive bacteria.

[0019] Figure 6 Detection of quercetin cytotoxicity against A549 cells.

[0020] Figure 7 Detection of quercetin cytotoxicity against J774A.1 cells.

[0021] Figure 8 The protective effect of quercetin combined with metacycline on infected mice. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] This invention first confirmed, through enzyme activity inhibition assays, minimum inhibitory concentration (MIC) assays, and time-bacterial killing curve assays, that quercetin significantly enhances the antibacterial activity of tetracycline antibiotics (tigecycline, minocycline, methacycline, and tetracycline) against Tet-X4-positive pathogens by activating the hydrolytic enzyme activity of Tet-X4. Further, through live-cell assays, it was determined that quercetin at effective synergistic concentrations had no significant toxic effects on A549 and J774A.1 cells. Finally, through systemic infection experiments in mice, it was confirmed that quercetin can significantly enhance the therapeutic effects of tetracycline antibiotics.

[0024] Example 1: Detection of quercetin's inhibition of Tet-X4 enzyme activity An inhibitor screening platform was established based on the principle of color and absorbance changes in tetracycline degradation by Tet-X4. Tet-X4 protein was co-incubated with quercetin at concentrations of 0 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, and 64 μg / mL, and then tetracycline and TAPS (Mg) were added. 2+ ) and NADPH, OD measured 400 nm The changes were carried out; the effect of the activity inhibitor on the kinetics of Tet-X4 enzyme was further analyzed by enzyme activity inhibition assay.

[0025] The test results are as follows Figure 1 As shown, the ability of Tet-X4 to hydrolyze tetracycline gradually decreases with increasing quercetin concentration. When the quercetin concentration is ≥16 μg / mL, its inhibition rate against Tet-X4 reaches more than 50%, indicating that low doses of quercetin can effectively inhibit the activity of Tet-X4, which may restore the in vitro and in vivo antibacterial activity of tetracycline antibiotics against Tet-X4 positive bacteria.

[0026] Example 2 Minimum Inhibitory Concentration Test The checkerboard method-microbroth dilution method was used to determine the minimum inhibitory concentration (MIC) of quercetin combined with tetracycline antibiotics (including tigecycline, minocycline, methacycline, and tetracycline) against Tet-X4-producing *Escherichia coli* and Tet-X4-producing *Klebsiella pneumoniae*. After continuous static incubation at 37°C for 24 hours, the results were observed and the synergistic inhibition index (FIC) was calculated. FIC = MIC (combined antibiotic concentration) / MIC (antibiotic single concentration) + MIC (combined quercetin concentration) / MIC (quercetin single concentration). The results are shown in Table 1.

[0027] Table 1. MIC and FIC values ​​of quercetin combined with different tetracycline antibiotics against different bacteria.

[0028] Note: Bold text indicates synergistic effect; the MIC value of quercetin against Escherichia coli and Klebsiella pneumoniae is 256 μg / mL, and the optimal combined concentration is 64 μg / mL; FICI < 0.5.

[0029] This study selected representative first-generation (tetracycline), second-generation (metazidine and minocycline), and third-generation (tigecycline) tetracycline antibiotics to test their synergistic effect with quercetin against Tet-X4-producing pathogens. As shown in Table 1, quercetin only showed significant synergistic antibacterial activity with tetracycline antibiotics against Tet-X4-producing pathogens (including Escherichia coli and Klebsiella pneumoniae) (FICI < 0.5), while no synergistic effect was observed against non-Tet-X4-producing pathogens (FICI > 0.5). Furthermore, quercetin did not exhibit synergistic effects with other types of antibiotics, suggesting that the synergistic effect of quercetin with tetracycline antibiotics is mainly due to quercetin's inhibitory effect on the Tet-X4 enzyme.

[0030] Example 3: Time-bacterial curve detection of quercetin combined with different tetracycline antibiotics Tet-X4-producing Escherichia coli cultured overnight E. coli The bacterial count of 47EC was adjusted to 1×10⁴ using a spectrophotometer. 8 CFUs / mL (OD 600nm =0.1), for later use. In sterile 96-well plates, a positive control group (no treatment), tetracycline antibiotic treatment groups (tigecycline, minocycline, methacycline, and tetracycline concentrations of 1 μg / mL, 16 μg / mL, 64 μg / mL, and 64 μg / mL, respectively), a quercetin treatment group (64 μg / mL), and a combined treatment group of both were set up. The bacterial culture was adjusted to a working bacterial count of 5 × 10⁻⁶. 5CFUs / mL. The culture was continuously incubated at 37℃ for 24 hours. At each time point, the bacterial suspension from the corresponding well was aspirated, serially diluted, and plated. On the second day, the number of single colonies formed on the agar plate at each time point for different groups was counted, and a time-sterilization curve was plotted.

[0031] The results are as follows Figure 2 As shown, tigecycline alone cannot completely kill Tet-X4-producing Escherichia coli in the culture medium. E. coli At 47EC, quercetin showed no significant bactericidal effect on the bacteria in the culture medium, but the combined use of tigecycline and quercetin completely killed all bacteria in the culture medium within 6 hours, indicating that tigecycline and quercetin have a significant synergistic bactericidal effect. Figures 3 to 5 As shown, minocycline, methacycline, or tetracycline alone cannot completely kill Escherichia coli in the culture medium. E. coli 47EC, and the combination of the two can completely kill Tet-X4-producing E. coli in the culture medium within 12 hours. E. coli 47EC indicates that different tetracycline antibiotics and quercetin have significant synergistic bactericidal effects against Tet-X4-producing pathogens.

[0032] Example 4: Quercetin cytotoxicity detection Tissue cells from different sources (human lung cancer epithelial cells A549 and mouse mononuclear macrophages J774A.1) were plated in 96-well cell culture plates, and different concentrations of quercetin (final concentrations of 0, 4, 8, 16, 32 and 64 μg / mL) were added. After culturing for 24 h, the supernatant was collected by centrifugation, and the lactate dehydrogenase content in the supernatant was detected using an LDH detection kit to analyze the survival rate of different cells.

[0033] The results are as follows Figure 6 and Figure 7 As shown, quercetin at concentrations within the detection range (≤64 μg / mL) showed no potential cytotoxicity to A549 and J774A.1 (LDH release <10%). This suggests that quercetin could be further investigated as a potential drug-active molecule in in vivo.

[0034] Example 5: Experimental therapeutic study of quercetin combined with methacycline on mice infected with Tet-X4 resistant bacteria. BALB / c mice were randomly divided into four groups (n=6) for a survival test. Each group received subcutaneous injections of methacycline (10 mg / kg), quercetin (50 mg / kg), a combination of methacycline and quercetin (10 mg / kg + 50 mg / kg), or an infection control group. Administered medications twice daily. All groups were also intraperitoneally infected with Tet-X4-producing resistant Escherichia coli. E. coli 47EC, challenge amount was 5.0×10 8CFU / mouse. Mice survival rate was observed until day 4.

[0035] The results are as follows Figure 8 As shown, quercetin combined with methacycline significantly increased the production of Tet-X4-producing Escherichia coli. E. coli Survival rate of mice infected with 47EC. This suggests that quercetin could be developed as a candidate drug for the prevention and treatment of Tet-X4 resistant bacterial infections.

[0036] The results from the above in vitro and in vivo experiments demonstrate that by inhibiting the biological activity of Tet-X4, the antibacterial activity of tetracycline antibiotics such as tigecycline or methacycline against Tet-X4-positive pathogens (including Escherichia coli and Klebsiella pneumoniae) can be significantly enhanced, thereby improving the survival rate of experimental animals infected with these pathogens. This discovery of the present invention has significant clinical application value and will provide an active ingredient and experimental basis for developing quercetin as a potentiator for tetracycline antibiotics.

[0037] The present invention has been further illustrated by the above embodiments, but is not intended to limit the invention in any way. Any modifications or alterations made to the present invention that are easily implemented by those skilled in the art without departing from the technical solutions of the present invention shall fall within the scope of the claims of the present invention.

Claims

1. Application of quercetin in the preparation of Tet-X4 enzyme inhibitors.

2. The application as described in claim 1, characterized in that, Quercetin can significantly inhibit the activity of Tet-X4 enzyme in hydrolyzing tetracycline antibiotics, thereby restoring the antibacterial activity of tetracycline antibiotics against Tet-X4 positive pathogens.

3. The application as described in claim 2, characterized in that, The Tet-X4 positive pathogens are Escherichia coli, Klebsiella pneumoniae, and Salmonella, which produce Tet-X4 enzymes; the tetracycline antibiotics are tigecycline, minocycline, methacycline, and tetracycline.

4. The application as described in claim 1, characterized in that, The specific application is: the combined use of quercetin and tetracycline antibiotics in the preparation of drugs for treating diseases caused by Tet-X4 positive pathogens.

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