Aromatic polyketone compound, preparation method and application

Abstract

The invention discloses an aromatic polyketone compound as well as a preparation method and application thereof. The compound GaB (3) has an effect of treating infection of multiple drug-resistant bacteria. The compound is obtained by being separated from a secondary metabolite of the symbiotic Streptomyces sp.QHH-9511 of licheniformis, and the compound 2, the compound 3, the compound 4 and the compound 5 are new compounds. According to the present invention, the research results show that the GaB can effectively sterilize a variety of drug-resistant bacteria (methicillin-resistant staphylococcus aureus, vancomycin-resistant enterococcus, carbapenem-resistant acinetobacter baumannii, multi-drug-resistant pseudomonas aeruginosa, multi-drug-resistant salmonella, and the like); in addition, the compound has the characteristics of broad-spectrum sterilization, biofilm formation inhibition and low-frequency drug resistance, and can be combined with other antibiotics to cope with super bacteria. Mechanism research shows that GaB is combined with glucosamine-6-phosphate synthetase (GlmS) to prevent synthesis of cell walls so as to play an antibacterial role, and a plurality of constructed living body models show that the compound can effectively reduce the content of bacteria in a body and promote wound repair and has in-vivo medication safety. The research result shows that the GaB can be used as a safe and effective novel pilot natural active small molecule for resisting infection of multiple drug-resistant bacteria.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and relates to an aromatic polyketide compound, a preparation method and application, in particular to secondary metabolites extracted from fermentation broth of a strain Streptomyces QHH-9511, a preparation method thereof and application in anti-multiple drug-resistant bacterial infection. BACKGROUND

[0002] Since the discovery of penicillin in 1928, researchers have developed more than 20 antibiotics with innovative structures in the following 30 years. However, since the 1980s, the development of new antibacterial drugs has been slow, almost at a standstill, which makes us have to rely too much on existing antibiotic resources. Bacteria have evolved a variety of drug resistance mechanisms, including reducing cell membrane permeability, using efflux pumps, modifying or degrading drugs through enzymes, changing antibiotic targets, and forming biofilms, almost all known antibiotics are affected to varying degrees. The World Health Organization (WHO) has listed bacterial drug resistance as one of the top ten public health threats facing humanity. According to a study recently published in The Lancet, antimicrobial drug resistance is expected to directly cause more than 39 million deaths and be associated with 169 million deaths between 2025 and 2050. QYResearch research shows that the global market size of methicillin-resistant Staphylococcus aureus (MRSA) treatment was about 3.815 billion US dollars in 2023, and is expected to reach 6.154 billion US dollars in 2030. Klein et al. (2024) counted the total consumption of antibiotics in 67 countries, which increased from 29.5 billion to 34.3 billion from 2016 to 2023, an increase of 16.3%. In the face of the rapid spread of multi-drug resistant bacteria, a serious threat to global public health, the key to solving this problem is the discovery of effective new antibiotics to treat modern drug-resistant bacteria, which makes it essential to develop antibiotics with broad-spectrum activity and low frequency of resistance. Therefore, finding natural small molecules as antibiotic candidate drugs has attracted great interest from researchers. SUMMARY

[0003] The application provides an aromatic polyketide compound, a preparation method and application, in particular to an aromatic polyketide compound and an effect of the aromatic polyketide compound in resisting multiple drug-resistant bacterial infection.

[0004] Specific schemes include:

[0005] The aromatic polyketide compound includes:

[0006] The aromatic polyketide compound includes the following compound 1, compound 2, compound 3 or compound 4:

[0007]

[0008] The preparation method of the aromatic polyketide compound is a method for preparing the aromatic polyketide compound isolated from the secondary metabolites of lichen symbiotic Streptomyces sp. QHH-9511.

[0009] The preparation method of the aromatic polyketide compound is a method for preparing the aromatic polyketide compound isolated from the ethyl acetate phase of the fermentation liquor of Streptomyces sp. QHH-9511.

[0010] The preparation method of the aromatic polyketide compound specifically comprises the following steps:

[0011] The strain Streptomyces sp. QHH-9511 is fermented in a modified yeast extract liquid medium at 28°C for 8 days, and then an extract is obtained by extraction with ethyl acetate;

[0012] The extract is separated by normal-phase silica gel column chromatography, gradient elution is performed with dichloromethane-methanol, and the gradient elution conditions are as follows: v / v, 100:0→100:8; four components Fr.1-Fr.4 are obtained; components Fr.2 and Fr.3 are separated and purified by reversed-phase RP-C 18 column MeOH-H2O elution with v / v 30%→100% to obtain effective components Fr.2.1-Fr.2.2 and Fr.3.1-Fr.3.2, and then gel column Sephadex LH-20 elution with MeOH solvent and semi-preparative HPLC separation and purification at MeCN-H2O volume concentrations of 40%-80% to obtain compounds 1-4.

[0013] Alternatively, the modified yeast extract liquid medium is as follows: peptone 2g, FeSO4·7H2O 0.1g, KBr 0.1g, yeast extract 4g, soluble starch 10g, CaCO3 1g, and water 1L.

[0014] The aromatic polyketide compound is used for preparing an application of an anti-Gram-positive bacteria and / or Gram-negative bacteria drug.

[0015] The aromatic polyketide compound is used for preparing an application of an anti-multiple drug-resistant bacteria infection drug.

[0016] The aromatic polyketide compound is used for preparing an application of a drug for treating meningitis, abdominal cavity infection and / or skin infection caused by multiple drug-resistant bacteria.

[0017] A drug for treating multiple drug-resistant bacteria infection, wherein the drug contains the aromatic polyketide compound.

[0018] An antibiotic, wherein the drug contains the aromatic polyketide compound described in this invention.

[0019] The advantages of this invention are:

[0020] The compounds of this invention were isolated from the secondary metabolites of the lichen symbiotic *Streptomyces* sp. QHH-9511, and compounds 2-4 are novel compounds. Studies have shown that GaB is effective against a variety of drug-resistant bacteria (methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, multidrug-resistant Acinetobacter baumannii, multidrug-resistant Pseudomonas aeruginosa, multidrug-resistant Salmonella, etc.). Furthermore, it exhibits broad-spectrum bactericidal activity, inhibition of biofilm formation, low-frequency resistance, and the ability to be used in combination with other antibiotics to combat "superbugs." Mechanistic studies have shown that GaB exerts its antibacterial effect by binding to glucosamine-6-phosphate synthase (GlmS) to prevent cell wall synthesis. Multiple in vivo models demonstrate that this compound can effectively reduce in vivo bacterial content, promote wound healing, and has in vivo safety. These findings indicate that GaB can serve as a safe and effective novel lead natural active small molecule for combating multidrug-resistant bacterial infections. Attached Figure Description

[0021] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings:

[0022] Figure 1 This study investigated the in vitro antibacterial activity of GaB. (A) Determination of GaB antibacterial activity using the agar dilution method; (B) Determination of GaB antibacterial activity using the inhibition zone method; (C) Growth curves of drug-resistant bacteria USA300 treated with different concentrations of GaB; (D) Time-dependent killing curves of drug-resistant bacteria USA300 treated with different concentrations of GaB; (E) Effect of different pH environments on the antibacterial effect of GaB; (F) Effect of different concentrations of GaB on biofilm formation; (G) Detection of the synergistic antibacterial effect of GaB and ampicillin.

[0023] Figure 2 To detect the resistance of drug-resistant bacteria USA300 to GaB. (A) Schematic diagram of the resistance test protocol; (B) Subculturing of drug-resistant bacteria USA300 for 20 consecutive generations at concentrations of GaB, norfloxacin (NFX), and ampicillin (AMP) at 0.5×MIC; (C) Inhibitory activity of GaB against norfloxacin-resistant USA300; (D) Inhibitory activity of GaB against ampicillin-resistant USA300. Data are expressed as mean ± SD (n=3), ***p<0.001, **p<0.01 and *p<0.05.

[0024] Figure 3To investigate the antibacterial mechanism of GaB against USA300. (A) Representative images from scanning electron microscopy (scale bar: 4.0 μm) and transmission electron microscopy (scale bar: 500 nm) before and after GaB treatment; (B) DAPI / PI fluorescence staining; (C) Visual diagram of GaB-GlmS (PDB: 4S1W) molecular docking; (D) GlmS-mediated bacterial cell wall biosynthesis; (E) Protein leakage; (F) ROS production; (G) Effect of GaB on GlmS enzyme activity; (H) Effect of adding G-6-F after GaB treatment on the growth of USA300. Data are expressed as mean ± SD (n = 3), ***p < 0.001, **p < 0.01 and *p < 0.05.

[0025] Figure 4 Safety assessment of compound GaB. (A) In vitro hemolytic activity assay of GaB. The hemolytic rate of mouse hemolysin cells was determined using the micro-dilution method, with Triton X-100 as the control group; (B) Cytotoxicity of GaB to five mammalian cell lines. MIA PaCa-2, H6C7, AGS, B16, and WPMY-1 cells were treated with different concentrations of GaB for 24 h, and the IC50 was determined. 50 Values; (CE) Acute toxicity test. Mice were intraperitoneally injected three times within 24 hours with 0.2 mL GaB (0.8 mg / kg), GaB (1.6 mg / kg), vancomycin (1.6 mg / kg), or PBS. (C) Changes in body weight of mice within 7 days after drug administration; (D) Histopathological evaluation of liver, kidney, and heart tissues of mice after drug administration (scale bar: 100 μm); (E) Content of white blood cells (WBCs), neutrophils (Gran#), lymphocytes (Lymoh#), hemoglobin (HGB), platelets (PLT), and red blood cells (RBCs) in the blood of mice after drug administration. Data are expressed as mean ± SD (n = 3), ***p < 0.001, **p < 0.01, and *p < 0.05.

[0026] Figure 5 To illustrate the in vivo efficacy of GaB against USA300 infection. (A) Schematic diagram of three in vivo infection experimental protocols; (B) Survival rate of G. melonella larvae infected with USA300 after treatment with different drug concentrations; (C) Bacterial content in G. melonella larvae; (D) Degree of skin wound healing in mice within 14 days; (E) Bacterial content in liver, heart, spleen, and kidney tissues of mice; (F) Illustration of skin wound healing in mice within 14 days; (G) Pathological evaluation of liver, kidney, and heart tissues of mice after drug administration (scale bar: 100 μm). Data are expressed as mean ± SD (n = 3), ***p < 0.001, **p < 0.01, and *p < 0.05. Detailed Implementation

[0027] This invention provides aromatic polyketide compounds and their effects against multidrug-resistant bacterial infections.

[0028] The structural formulas of the aromatic polyketide compounds of this invention are shown below:

[0029]

[0030] Physicochemical properties of compound (1) of the present invention: C 28 H 32 O 13 Red powder; (c=0.05,MeOH); ESI-MSm / z577.55[M+H] + The structure is consistent with that of Granaticinic acid methyl ester reported in the literature (Deng MR, Li Y, Luo X, Zheng XL, Chen Y, Zhang YL, Zhang W, Zhou H, Zhu H. Discovery of Mycothiogranaticins from Streptomyces vietnamensis GIMV4.0001 and the Regulatory Effect of Mycothiol on the Granaticin Biosynthesis. FrontChem. 2021.9:802279), therefore compound 1 is identified as Granaticinic acid B.

[0031] Physicochemical properties of compound (2) of the present invention: C 29 H 34 O 13 Red powder; [α] D 20 -163.5(c 0.05,MeOH); HRESIMSm / z613.1895[M+Na] + (calcd.for NaC 29 H 34 O 13 ,613.1895); 1 H-NMR spectrum and 13 The C-NMR spectral data are shown in Table 1.

[0032] Physicochemical properties of compound (3) of the present invention: C 30 H 32 O 13 Red powder; [α] D 20-169.7(c 0.05,MeOH); HRESIMS m / z623.1735[M+Na] + calcd.623.1735; 1 H-NMR spectrum and 13 The C-NMR spectral data are shown in Table 1.

[0033] Physicochemical properties of compound (4) of the present invention: C 28 H 28 O 12 Red powder; [α] D 20 -174.9(c 0.05,MeOH); HRESIMS m / z579.1473[M+Na-H2O] + calcd.579.1473; 1 H-NMR spectrum and 13 The C-NMR spectral data are shown in Table 1.

[0034] The method for preparing aromatic polyketide compounds of the present invention involves isolating them from the fermentation broth of Streptomyces QHH-9511.

[0035] Aromatic polyketides were isolated from the ethyl acetate phase of the Streptomyces QHH-9511 fermentation broth.

[0036] Specifically, the extract was obtained by fermenting strain Streptomyces sp. QHH-9511 at 28°C on a shaker (120 rpm) for 8 days in a modified yeast extract liquid medium, followed by extraction with ethyl acetate.

[0037] The extract was separated by normal-phase silica gel column chromatography with dichloromethane-methanol gradient elution under the following conditions: v / v, 100:0 → 100:8; four fractions Fr.1-Fr.4 were obtained; fractions Fr.2 and Fr.3 were separated by reverse-phase RP-C chromatography. 18 The effective components Fr.2.1-Fr.2.2 and Fr.3.1-Fr.3.2 were obtained by elution with MeOH-H2O column at a v / v ratio of 30% to 100%. Then, they were separated and purified by gel column Sephadex LH-20 with MeOH solvent and semi-preparative HPLC at a MeCN-H2O volume concentration of 40%-80% to obtain compounds 1-4.

[0038] The aromatic polyketide compounds of the present invention are used in the preparation of anti-Gram-positive and Gram-negative bacteria.

[0039] The aromatic polyketide compounds of the present invention are used in the preparation of drugs against multidrug-resistant bacterial infections.

[0040] The aromatic polyketide compounds of the present invention are used to prepare treatments for various diseases caused by different pathogens, such as skin infections caused by Staphylococcus aureus, meningitis caused by multidrug-resistant Acinetobacter baumannii, and sepsis caused by multidrug-resistant Pseudomonas aeruginosa.

[0041] A medicine for treating multidrug-resistant bacterial infections, the medicine containing the aromatic polyketide compound of the present invention.

[0042] The present invention also provides a method for preparing the compound, comprising the following steps:

[0043] (1) Seed culture: The strain Streptomyces QHH-9511 was inoculated from the seed tube in the -80℃ refrigerator into Gao's No. 1 medium and placed on a shaker at 28℃ and 120r / min for three days to obtain the seed culture.

[0044] (2) Expanded culture: Take 5 mL of seed liquid and inoculate it into a culture flask (1000 mL) of 400 mL modified yeast extract medium. Divide the flask into 140 flasks and place them in a shaker in a fermentation room at a constant temperature of 28℃ for 8 days.

[0045] (3) Obtaining secondary metabolites: Extract the fermentation broth three times with twice the volume of ethyl acetate. Concentrate again to obtain the extract.

[0046] (4) Separation and purification of compounds: The extract was subjected to normal phase silica gel column chromatography (300-400 mesh) and gradient elution with petroleum ether-ethyl acetate solution (100:0-100:100, v / v) to obtain four fractions Fr.1-Fr.4.

[0047] (5) Preparation of compound 1: Component Fr.2 was prepared by reverse RP-C 18 The effective components Fr.2.1-Fr.2.2 were obtained by elution with MeOH-H2O column at v / v, 30%→100%. Among them, component Fr.2.1 was subjected to gel column Sephadex LH-20 with MeOH as mobile phase to obtain compound 1 (820 mg).

[0048] (6) Preparation of compound 2: Component Fr.3 was prepared by reverse RP-C 18 The active components Fr.3.1-Fr.3.2 were obtained by elution with MeOH-H2O column at v / v, 30%→100%. Fr.3.1 was further purified by semi-preparative HPLC under 55% MeCN (0.2‰ TFA) to obtain compound 2 (t R =27min, 120mg);

[0049] (7) Preparation of compound 3: Component Fr.2.2 was eluted on a Sephadex LH-20 gel column with MeOH as the mobile phase, and then purified by semi-preparative HPLC at 50% (MeCN 0.2‰ TFA) to obtain compound 3 (t R =31min, 182mg);

[0050] (8) Preparation of compound 4: Fr3.2 was purified by semi-preparative HPLC under 58% MeCN (0.2‰ TFA) to obtain compound 4 (t R =25min, 22mg).

[0051] Structural analysis of compound 2: High-resolution mass spectrometry analysis of compound 2 revealed a [M+Na]+ peak at m / z 613.1895, confirming the molecular formula of compound 2 as C. 29 H 34 O 13 After comparing the NMR data with those of compound 1, it was found that they have almost the same planar structure. The only difference is that compound 2 has an additional methoxy group (δ-hydroxyl group) located at C-23. H / δ C (3.65 / 52.0), this conclusion is confirmed by the HMBC correlation from H3-23 to C-1. Since compounds 2 and 1 have the same biosynthetic pathway, similar ECD curves and NOESY correlation signals, compounds 2 and 1 are considered to have the same absolute configurations 3S, 4S, 15S, 17R, 19R, 20R and 21R and are named neogranaticin A.

[0052] Structural analysis of compound 3: Compound 3 was determined by high-resolution mass spectrometry to have an m / z of 431.1335 [M+Na-H2O]. + The peaks confirmed that the molecular formula of compound 3 is C3. 30 H 34 O 14 . 1 H and 13 The 1 / 2C NMR data showed that it was very similar to compound 1, except that it had an additional methyl ester group [COO-7'(δ)]. C 170.9) and CH3-8'(δ) H 2.10,δ C[21.2], linked to the C-4' hydroxyl group on α-L-rhodinose, as confirmed by correlations in the HMBC spectrum from H-4' to C-7' and from H3-8' to C-7'. Considering that compounds 3 and 1 have similar NMR data, coupling constants, NOESY correlations, and ECD curves, their absolute configurations should be consistent as 3S, 4S, 15S, 17R, 19R, 20R, and 21R, and they should be named neogranaticin B (GaB).

[0053] Compound 4 was determined by high-resolution mass spectrometry to have an m / z of 579.1473 [M+Na-H2O]. + The peaks confirmed that compound 4 has the molecular formula C. 28 H 30 O 13 . 1 H and 13 The C10 NMR data show that it is very similar to compound 1. The difference is that the hydroxyl group at C-4' of α-L-rhodinose in compound 4 is oxidized to a carbonyl group. The HMBC spectrum shows that from H2-3' and H-5' to the carbonyl C-4' (δ) C The correlation of 209.9) confirms this. Considering that compounds 4 and 1 have similar NMR data, coupling constants, NOESY correlations and ECD curves, their absolute configurations should be consistent as 3S, 4S, 15S, 17R, 19R, 20R and 21R and named neogranaticin C.

[0054] Table 1: Compounds 2-4 1 HNMR (400MHz, Methanol-d4) and 13 CNMR (100MHz, Methanol-d4) data (Acetone-d6)

[0055]

[0056]

[0057] Example 1: Preparation of compounds 1-4

[0058] The strain Streptomyces QHH-9511 (currently deposited at the College of Chemistry and Pharmacy, Northwest A&F University, see the paper "Genomic and Metabolite Profiling Reveal a Novel StreptomycesStrain, QHH-9511, from the Qinghai-Xizang Plateau") was inoculated from a preservative tube stored at -80℃ into Gao's No. 1 medium and activated for three days on a shaker at 28℃ and 120 r / min to obtain a seed culture. Then, 5 mL of the seed culture was inoculated into 1000 mL culture flasks containing 400 mL of modified yeast extract medium, for a total of 140 flasks, and cultured on a shaker in a fermentation room at a constant temperature of 28℃ for 8 days. After fermentation was complete, the extract was extracted three times with twice the volume of ethyl acetate and then concentrated to obtain 34.5g of extract. The modified yeast extract liquid culture medium was: 2g peptone, 0.1g FeSO4·7H2O, 0.1g KBr, 4g yeast extract, 10g soluble starch, 1g CaCO3, and 1L water.

[0059] The extract was separated by normal-phase silica gel column chromatography with a dichloromethane-methanol gradient elution under the following conditions: v / v, 100:0 → 100:8; four fractions Fr.1-Fr.4 were obtained; fraction Fr.2 was separated by reverse-phase RP-C chromatography. 18 The effective fractions Fr.2.1-Fr.2.2 were obtained by elution with a MeOH-H2O column at v / v, 30%→100%. Fraction Fr.2.1 was further purified by a Sephadex LH-20 gel column with MeOH as the mobile phase to obtain compound 1 (820 mg). Fraction Fr.2.2 was eluted by a Sephadex LH-20 gel column with MeOH as the mobile phase and then purified by semi-preparative HPLC at 50% (MeCN 0.2‰ TFA) to obtain compound 3 (t). R =31 min, 182 mg); component Fr.3 via reverse-phase RP-C 18 The active components Fr.3.1-Fr.3.2 were obtained by elution with MeOH-H2O column at v / v, 30%→100%. Fr.3.1 was further purified by semi-preparative HPLC under 55% MeCN (0.2‰ TFA) to obtain compound 2 (t R =27 min, 120 mg), Fr3.2 was separated and purified by semi-preparative HPLC under 58% MeCN (0.2‰ TFA) to obtain compound 4 (t R =25min, 22mg).

[0060] Example 2: Pharmacological activity of the compound

[0061] Test methods

[0062] 1. Antimicrobial susceptibility testing

[0063] The minimum inhibitory concentration (MIC) of the compound was determined using the microbroth dilution method. The compound was serially diluted twofold with the antibiotic in broth medium to obtain different concentrations ranging from 0.6 to 2560 μg / mL. 10 μL of each concentration was added to a 96-well plate containing 190 μL of bacterial culture per well, resulting in final drug concentrations ranging from 0.03 to 128 μg / mL and bacterial concentrations of 5 × 10⁻⁶. 5 CFU / mL, mixed thoroughly, and incubated at 37℃ for 18 hours. MIC is the lowest drug concentration at which the wells become clear and transparent, completely inhibiting bacterial growth. The inhibition zone method is used, which determines the antibacterial potency of the test drug by observing the diffusion of the drug in the agar plate, where the surrounding bacteria are inhibited and a clear zone is formed. The agar dilution method is used, where different concentrations of drug are added to dissolved agar medium at approximately 55℃, poured into plates, allowed to solidify, and then spread onto test bacterial suspensions for observation of bacterial growth. Additionally, the pH of the medium is adjusted to 5–10 with HCl or NaOH as needed to observe the growth of test bacteria in media containing the drug at different pH values.

[0064] 2. Growth curve

[0065] Adjust the concentration of the drug-resistant bacteria USA300 to 1×10⁻⁶. 7 CFU / mL of GaB was added to achieve final drug concentrations of 0.5×MIC, 1×MIC, 2×MIC, and 4×MIC, along with a blank control group. The bacteria were cultured in a 37℃ shaker for 24 h. Every 2 h, the absorbance at OD600 nm was measured using an Infinite E Plex microplate reader (TECAN, Switzerland) to obtain growth curves for USA300 at different drug concentrations. In the survival rescue experiment, 1 mM fructose-6-phosphate (G-6-F) was added to the bacterial suspension with an OD600 nm of 0.5. Simultaneously, a bacterial suspension incubated with 2×MIC GaB was used as a control group.

[0066] 3. Time-sterilization curve

[0067] Adjust the concentration of the drug-resistant bacteria USA300 to 1×10⁻⁶. 7 CFU / mL, different concentrations of GaB were added to achieve final drug concentrations of 0.5×MIC, 1×MIC, 2×MIC, and 4×MIC, along with a blank control group. The mixtures were incubated in a 37℃ constant temperature shaker for 24 h, with samples taken every 2 h. Each sample was then serially diluted 10-fold to 10⁻⁶ CFU / mL. 8times. Absorb 100 μL and spread it on an agar plate. After 18 h, count the colonies (expressed as CFU / mL). Plot a time-colony count logarithmic curve with the logarithm of CFU / mL as the ordinate and time as the abscissa. The bactericidal rate and duration of action of different concentrations of the drug can be compared through this curve.

[0068] 4. FIC combination therapy - Evaluation of antibacterial drug interactions

[0069] The microdilution checkerboard method was used to determine the inhibitory effect of the combination of compound GaB and ampicillin on the test bacteria, so as to judge whether there is a synergistic effect, additive effect or antagonistic effect between the two. Based on the minimum inhibitory concentration MIC measured previously, the MIC of the compound used alone and in combination with antibiotics was measured, and the FIC index was calculated. That is, compound GaB was serially diluted two-fold along the horizontal row of the 96-well plate, and ampicillin was serially diluted two-fold along the vertical row and added to the wells containing the bacterial solution in turn. After culturing for 18 h, the minimum concentration combination without bacterial growth is the MIC of the two drugs in combination. The FIC index is calculated according to the following formula: FICindex = MIC AB / MIC A +MIC BA / MIC B

[0070] Where MICA is the MIC of drug A used alone; MICAB is the MIC of drugs A and B in combination; MICB is the MIC of drug B used alone; MICBA is the MIC of drugs A and B in combination. When FICI ≤ 0.5, the mode of action of the two drugs is synergistic; when 0.5 < FICI ≤ 1, the mode of action of the two drugs is additive; when 1 < FICI ≤ 2, the mode of action of the two drugs is irrelevant; when FICI > 2, the mode of action of the two drugs is antagonistic.

[0071] 5. Biofilm formation inhibition experiment

[0072] Add the bacterial solution with a concentration of 1×10 8 CFU / mL into a 96-well plate, and then add compound GaB to make its final concentrations 1 / 4×MIC, 1 / 2×MIC and 1×MIC respectively. The bacterial solution without adding the compound is used as the positive control. After incubating in a 37 °C incubator for 48 h to form a biofilm, aspirate the bacterial solution in the wells, gently wash the remaining biofilm with PBS twice, then fix the biofilm with methanol for 15 min and aspirate the methanol, stain the biofilm with 0.1% crystal violet for 15 min, and wash it three times with PBS to remove the excess crystal violet. Finally, dissolve the crystal violet with 95% ethanol, and measure the absorbance at 595 nm using an infinite E Plex microplate reader (TECAN, Switzerland).

[0073] 6. Bacterial drug resistance detection

[0074] The resistance of drug-resistant bacteria USA300 to compound GaB was determined using a drug resistance assay, with norfloxacin and ampicillin serving as positive controls. First, the inhibitory concentrations (MICs) of GaB, norfloxacin, and ampicillin against MRSA were determined. Then, a concentration of the test compound at 1 / 2 × MIC was added to the bacterial culture and incubated at 37°C for 24 hours. Subsequently, the culture was continuously subcultured daily to media containing progressively increasing concentrations of the compound, with a drug-free control included. The MICs were re-determined using the microbroth dilution method, and changes in MIC values ​​were recorded. After 20 generations of continuous subculturing, the rate of resistance development was assessed by comparing the fold increase in MIC (experimental MIC / initial MIC) between the experimental and control groups.

[0075] 7. DAPI / PI fluorescent staining

[0076] MRSA strains were inoculated into broth medium and cultured on a shaker at 37°C and 120 rpm for 12 h. The cells were then obtained by centrifugation at 5000 rpm, washed twice with PBS, and resuspended in PBS to a bacterial concentration of 1 × 10⁻⁶. 9 CFU / mL. GaB at final concentrations of 4×MIC and 8×MIC was added to the bacterial suspension and incubated at 37°C for 2 h. The cells were obtained by centrifugation, washed twice with PBS, and resuspended in 20 μL of DAPI (10 μg / mL) and 20 μL of PI (20 μg / mL) solution. The suspension was incubated in the dark for 30 min, washed twice with PBS, and resuspended in 200 μL of PBS. Finally, the bacterial suspension was dropped onto a glass slide, covered with a slide, and photographed using a LEICA TCS SP8 laser confocal microscope (Leica, Germany).

[0077] 8. Detection of protein leakage and reactive oxygen species (ROS) levels

[0078] USA300 was cultured overnight on a shaker (200 rpm, 37°C), and the OD600nm was adjusted to 0.5. Different concentrations of GaB (2×MIC, 4×MIC, and 8×MIC) were added to the bacterial suspension and incubated at 37°C for 3 h. Intracellular protein leakage was measured according to the instructions of the BCA Protein Assay Kit (Beyotime, China). ROS accumulation in bacteria was measured using a ROS Assay Kit (Beyotime, China) with the DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) fluorescent probe, following the manufacturer's instructions. 10 μM of DCFH-DA was added to the bacterial suspension and incubated at 37°C for 30 min. The bacterial suspension was washed with PBS to remove the fluorescent dye, and then different concentrations of GaB were added to 190 μL of bacterial suspension, resulting in final concentrations of 2×MIC, 4×MIC, and 8×MIC. An equal volume of PBS was used as a blank control. After incubation for 20 minutes, the fluorescence intensity was measured using an Infinite M200 microplate reader (Tecan, Switzerland).

[0079] 9. Scanning electron microscope

[0080] Adjust the bacterial concentration to 1×10⁻⁶ 8 CFU / mL was added, followed by GaB (4×MIC), and incubated on a shaker at 37°C and 120 rpm for 3 hours. Bacterial cells were obtained by centrifugation at 5000 rpm, washed twice with PBS, and fixed with 2.5% glutaraldehyde. Simultaneously, a silicon wafer was placed in the bacterial solution to allow natural adsorption and fixation onto the wafer. The wafer was then incubated overnight at 4°C, followed by three washes with PBS for 10 minutes each. Next, the wafer was dehydrated three times sequentially with 30%, 50%, 70%, 80%, and 90% ethanol for 30 minutes each time. After CO2 drying, the silicon wafer containing the bacterial cells was adhered to conductive adhesive, sputtered with gold, and photographed using a SU8600 cold field emission scanning electron microscope (Hitachi, Japan).

[0081] 10. Transmission electron microscopy

[0082] Bacterial culture cultured on a shaker for 12 h was added to GaB (4×MIC) and incubated on a shaker at 37℃ and 120 rpm for 3 h. Cells were obtained by centrifugation at 5000 rpm, washed twice with PBS, fixed overnight with 2.5% glutaraldehyde at 4℃, then fixed with 1% osmium tetroxide for 3 h, and washed three times with PBS for 15 min each time. Subsequently, the cells were dehydrated twice with 30%, 50%, 70%, 80%, 90%, and 100% ethanol, 10 min each time. The cells were then infiltrated with 25% embedding medium for 2 h, 50% embedding medium for 8 h, 75% embedding medium for 12 h, and pure embedding medium twice, 24 h each time. After embedding and solidification, sections were prepared using an ultramicrotome, and finally photographed using an HT7800 transmission electron microscope (Hitachi, Japan).

[0083] 11. Molecular docking

[0084] To more accurately identify the protein targets of GaB, multiple literature reviews were consulted, and commonly discussed antibacterial targets were docked one by one. The target with the lowest binding energy was selected, and the three-dimensional crystal structures of proteins SaFabI (PDB: 4FS3), NDH-2 (PDB: 5NA1), GyrB (PDB: 3TT2), GlmS (PDB: 4S1W), DHFR (PDB: 3IX9), PGP (PDB: 5JWY), and CLPP (PDB: 7WID) were docked with the standard three-dimensional structure of GaB (mol2 format) using the CB-Dock2 online molecular docking tool. After obtaining the target with the lowest binding energy, GlmS, the binding mode between the protein and ligand molecules was visualized using Discovery Studio software. The molecular docking results were evaluated in terms of interaction sites and interaction force types.

[0085] 12. GlmS enzyme activity assay

[0086] The procedure was performed according to the instructions for the 6-phosphate glucosamine synthase kit (Grace Biotech, China). Sample pretreatment: Different concentrations of GaB were added to USA300 bacterial culture and incubated at 37°C for 6 hours using a shaker. Bacteria were collected, centrifuged to obtain a bacterial pellet, and approximately 5 × 10⁻⁶ cells were collected. 6 Add one bacterium to 1 mL of extraction solution and sonicate to disrupt the bacteria under ice bath conditions. Repeat this process 30 times, sonicating for 3 seconds followed by a 10-second pause. Then centrifuge at 12000 rpm for 10 min at 4℃, collect the supernatant, and place it on ice. Detection procedure: Follow the instructions, adding reagents sequentially and reacting at the appropriate temperatures. Finally, transfer the liquid to a 1 mL cuvette and measure the absorbance (A) at 450 nm using a UV-1780 UV-Vis spectrophotometer (Shimadzu, China). Enzyme activity definition: One unit of enzyme activity is defined as the generation of 1 nmol of glutamate per milliliter of liquid per hour. Where ΔA = Aassay - Acontrol.

[0087] GlmS(nmol / h / mL)=[(ΔA+0.0005)÷14.453]×V2×103÷V1÷T=346×(ΔA+0.0005), V1=sample volume added, 0.24mL; V2=total reaction volume, 0.6mL; T=reaction time, 30min=0.5h.

[0088] 13. Drug safety assessment

[0089] (1) Hemolysis test

[0090] Fresh mouse blood was collected and centrifuged at 1500 rpm to obtain blood cells. The cells were then washed with 0.9% physiological saline until the supernatant was no longer red. The red blood cells were then resuspended in 0.9% physiological saline to prepare a 6% suspension. Using a micro-dilution method, compound GaB, a positive control (Triton-X-100), and a negative control (PBS, 0.01 M, pH 7.4) were sequentially added to 96-well plates containing 6% blood cells. The final concentration of GaB ranged from 0 to 1024 μg / mL, and the final concentration of Triton-X-100 ranged from 0 to 1%. The 96-well plates were then incubated at 37°C for 1 hour. After centrifugation at 1500 rpm for 2 minutes, 100 μL of the supernatant was transferred to a new 96-well plate, and the absorbance at OD540 nm was measured to quantitatively study hemolysis. The hemolysis rate was calculated using the following equation:

[0091]

[0092] (2) Cytotoxicity assay

[0093] The toxicity of compound GaB to mammalian cells was determined using the MTT assay. Five different cell lines—human pancreatic cancer cells (MIAPaCa-2), normal human pancreatic ductal epithelial cells (H6C7), human gastric adenocarcinoma cells (AGS), melanoma cells (B16), and human normal prostate stromal immortalized cells (WPMY-1)—were cultured at 1 × 10⁻⁶ cells per well. 4 Cells were seeded in 96-well plates and incubated overnight in a cell culture incubator. Then, they were treated with different concentrations of GaB for 24 hours. Next, 10 μL of MTT (5 mg / mL) solution was added to each well, and the plates were incubated at 37°C for 3 hours. The absorbance was measured at OD490 nm, and the IC50 of GaB for each cell type was calculated. 50 value.

[0094] (3) Acute toxicity test in mice

[0095] To investigate whether compounds GaB and vancomycin induce acute poisoning in mice, mice were divided into four groups (n=6 per group). Each group of mice received an intraperitoneal injection of 0.2 mL of GaB (0.8 mg / kg), GaB (1.6 mg / kg), vancomycin (1.6 mg / kg), or PBS every 8 hours, for a total of three injections. The mice were weighed daily to monitor their health. After 7 days, the mice were euthanized and dissected. Blood samples were collected for three-part differential hematologic analysis. Additionally, spleen, kidney, heart, and liver tissues were collected, fixed, sectioned, stained with hematoxylin and eosin (H&E), and scanned.

[0096] 14. Galleria mellonella infection model

[0097] G. melonella larvae (approximately 0.6 g each) were randomly divided into 6 groups (10 larvae per group), with 5 groups receiving an injection of 10 μL LUSA300 bacterial suspension (5 × 10⁻⁶) into the right posterior abdominal leg. 7 One group received 10 μL of PBS as a blank control, while another group received 10 μL of PBS as a blank control. After incubating at 37°C for 2 hours, four groups were randomly selected from the five groups that received bacterial inoculation and injected 10 μL of GaB (5, 10, 15 mg / kg) and 10 μL of vancomycin (10 mg / kg) into the left hind abdominal leg, respectively (drug group and positive control). The remaining two groups received 10 μL of PBS (negative control and blank control). The larvae were incubated at 37°C for 72 hours, and the survival of *G. mellonella* larvae was observed. Similarly, six additional groups of five larvae each were set up. Twenty-four hours after drug injection, the entire larvae were homogenized in PBS and then serially diluted 10-fold to 10-fold. 8 Double the volume, take 100 μL and spread it on an agar plate. After 18 h, count the colonies (expressed as Lg CFU / mL).

[0098] 15. Mouse model of skin wound infection

[0099] Mice were anesthetized by intraperitoneal injection of afodin tribromoethanol (20 mL / kg). The hair on the mice's backs was then removed using a hair removal device. A circular incision with a diameter of 10 mm was made on the back using surgical scissors. Following this, a solution containing 1×10... 8 100 μL of PBS containing CFU / mL bacterial solution was inoculated into each wound. On the second day post-infection, mice were divided into three groups of six, with wounds treated once daily for six consecutive days with 50 μL GaB (10 mg / kg), vancomycin (10 mg / kg), or PBS, respectively. Wounds were observed, photographed, and their size measured during the healing process.

[0100] 16. Mouse peritonitis-septicemia model

[0101] Mice were intraperitoneally injected with 0.2 mL of USA300 (5 × 10⁻⁶). 8 Two hours after infection, mice were randomly divided into 5 groups (n=10 per group) and intraperitoneally injected with 0.2 mL of GaB (0.2 mg / kg), GaB (0.4 mg / kg), GaB (0.8 mg / kg), vancomycin (0.8 mg / kg), and PBS, once every 8 hours for a total of three injections. On the third day post-infection, the mice were euthanized, and their hearts, livers, spleens, and kidneys were harvested and homogenized with PBS. The bacterial load in these organs was counted by colony counting. Tissues from each group were randomly selected, placed in fixative for sectioning, stained with hematoxylin and eosin (H&E), and scanned.

[0102] Test results

[0103] 1. In vitro antibacterial activity

[0104] First, the in vitro antibacterial activity of all compounds was evaluated using the broth microdilution method to determine their MICs against 8 Gram-positive and 11 Gram-negative bacteria. Retamoline (RPM) and ampicillin (AMP) were selected as positive controls. Compound 3 (GaB) had the lowest MIC (0.125 μg / mL) against USA300 among the pathogens tested; therefore, compound GaB was selected for further investigation of its antibacterial properties and mechanisms (details are shown in Table 2). The MBC values ​​of GaB against MRSA171, CMCC 44103, and MRSA USA300 were determined using the agar dilution method to be 32 μg / mL, 1 μg / mL, and 1 μg / mL, respectively. Figure 1 A; Furthermore, the inhibition zones showed that different concentrations of GaB inhibited the 12 strains to varying degrees, and the inhibition was dose-dependent. Figure 1 B); Compared with the control group, GaB concentration of 0.5×MIC did not affect the growth of USA300, while concentration of 1×MIC significantly delayed the growth of USA300. However, concentrations of 2×MIC and 4×MIC completely inhibited the growth of the strain. Figure 1 C). In addition, we monitored the survivability of USA300 exposed to different concentrations of GaB at different times (C). Figure 1 D) When the concentration is 1×MIC, it can basically inhibit the growth of USA300, with an initial bacterial count of 1×10⁻⁶. 7 At a concentration of CFU / mL, treatment with GaB at a concentration of 2×MIC essentially killed the bacteria within approximately 16 hours. We then adjusted the pH of the culture medium and investigated the effect of different acid-base conditions on the antibacterial activity of GaB. Figure 1E), the results showed that the antibacterial activity decreased rapidly with increasing pH. Bacterial biofilm (BBF) is an important indicator of bacterial resistance. It is a special microbial aggregate composed of bacteria and their secreted extracellular macromolecular polymers, possessing a unique spatial structure that can form an effective protective barrier against the bactericidal effects of antibiotics. Therefore, we tested whether GaB could inhibit bacterial biofilm formation, and the results were as follows. Figure 1 F indicates that at a concentration of 1×MIC, biofilm formation can be essentially completely inhibited. Furthermore, GaB can synergistically inhibit multiple pathogens with ampicillin. Figure 1 The lowest G)FIC value can reach 0.125. In summary, this indicates that GaB can effectively inhibit the growth of drug-resistant pathogens and is a promising natural small molecule that could become a candidate antibacterial drug.

[0105] Table 2. In vitro antibacterial activity (MIC) of compounds a (μg / mL)

[0106]

[0107] a. MIC was determined by three independent experiments; b. Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 12228; c. Staphylococcus aureus (S. aureus) ATCC 29213; d. Methicillin-resistant Staphylococcus aureus (MRSA) ATCC BAA-1717 (USA300); e. Methicillin-resistant Staphylococcus aureus (MRSA) 209; f. Methicillin-resistant Staphylococcus aureus (MRSA) 575; g. Methicillin-resistant Staphylococcus aureus (MRSA) 171; h. Vancomycin-resistant Enterococcus 1880; i. Vancomycin-resistant Enterococcus 1850; j. Vancomycin-resistant Enterococcus 1894; k. Escherichia coli (CMCC) 44103; l. Escherichia coli (CMCC) 25922; m. Acinetobacter baumannii (ATCC) 19606; n Acinetobacter baumannii 560; o Carbapenem-resistant Acinetobacter baumannii 178; p Acinetobacter baumannii 882; q Multidrug-resistant Pseudomonas aeruginosa 110; r Multidrug-resistant Pseudomonas aeruginosa 174; s Multidrug-resistant Pseudomonas aeruginosa 264; t Salmonella 315; u Multidrug-resistant Salmonella 242; v Ampicillin; w Retaparin

[0108] 2. GaB effectively controls drug resistance frequency.

[0109] Bacterial resistance is also an important indicator for assessing the clinical application potential of antimicrobial drugs. To evaluate whether USA300 would develop resistance to GaB, 20 consecutive passages were performed at a concentration of 0.5×MIC to test the resistance of USA300 to GaB, norfloxacin (NFX), and ampicillin (AMP). Figure 2A) Norfloxacin developed resistance after 3 generations of passages, and the MIC value increased 64-fold after 20 generations; ampicillin developed resistance after 2 generations, and the MIC value increased from 0.25 μg / mL to 128 μg / mL after 20 generations; while GaB began to develop resistance after 7 generations, and the MIC value increased 2-fold after 20 generations. Figure 2 B). Furthermore, GaB is effective against norfloxacin (B). Figure 2 C) and ampicillin ( Figure 2 D) The drug-resistant USA300 (after 20 generations) exhibited strong resistance to multidrug-resistant bacteria. These results indicate that GaB has the potential to prevent bacterial resistance and provides good resistance to some drug-resistant strains.

[0110] 3. Research on antibacterial mechanisms

[0111] First, we used SEM and TEM to observe the effects of GaB treatment on the external and internal morphological changes of bacteria. SEM results showed ( Figure 3 A) In the blank control group, USA300 bacteria remained morphologically intact and had smooth surfaces, while the bacteria in the GaB (4×MIC) treated group showed severe rupture, abnormally dividing into two halves. TEM results showed ( Figure 3 A) In the blank control group, the bacteria maintained intact internal morphology, with clearly visible cell walls and cell membranes. However, the cell walls of the bacteria in the GaB-treated group were significantly thinner than those in the blank group, affecting the synthesis of septal cell walls and leading to abnormal bacterial division. This indicates that GaB affects cell wall formation, thus causing bacterial death. Furthermore, we used the fluorescent dyes PI and DAPI to observe the damage to USA300 cells. DAPI can penetrate intact bacterial cell membranes and bind tightly to DNA in the cell nucleus, producing blue fluorescence. PI, on the other hand, can only penetrate damaged cell membranes and similarly bind to intracellular DNA, emitting red fluorescence. Figure 3 As shown in Figure B, the blank control group only emitted blue fluorescence, indicating that the bacteria remained morphologically intact. However, when GaB acted on the bacteria, the blue fluorescence weakened, and red fluorescence appeared simultaneously. Furthermore, the red fluorescence increased with increasing drug concentration, indicating that GaB can destroy bacteria in a dose-dependent manner, causing cell wall rupture, cell membrane damage, and ultimately bacterial death. Figure 3 As can be seen from E and F, the disruption of the bacterial cell wall, damage to the cell membrane, or changes in permeability can lead to the leakage of intracellular macromolecular proteins and the production of ROS in a dose-dependent manner. Excessive ROS is toxic to bacteria and accelerates bacterial death.

[0112] To identify targets for GaB, we performed molecular docking on seven commonly studied target proteins: SaFabI (PDB: 4FS3), NDH-2 (PDB: 5NA1), GyrB (PDB: 3TT2), GlmS (PDB: 4S1W), DHFR (PDB: 3IX9), PGP (PDB: 5JWY), and CLPP (PDB: 7WID). We ultimately screened for the protein with the lowest binding energy, -11.0 kJ / mol, which binds to GlmS. GaB can form four hydrogen bonds with the amino acid residues ARG462, ARG584, SER487, and ASP509 of GlmS. Figure 3 C) indicates a relatively close interaction between the ligand and receptor, with strong stability and affinity in their binding. Furthermore, studies on GlmS revealed that it catalyzes the conversion of fructose-6-phosphate (G-6-F) to glucosamine-6-phosphate (GlcN6P) in bacteria, a crucial step in peptidoglycan biosynthesis. GlcN6P produced by GlmS is an important precursor for the synthesis of UDP-GlcNAc, and UDP-GlcNAc is the glycosyl donor in peptidoglycan biosynthesis. Therefore, GlmS provides the necessary material basis for cell wall peptidoglycan synthesis by participating in the regulation of UDP-GlcNAc production. Figure 3 D). Inhibition of GlmS function leads to reduced UDP-GlcNAc production, which in turn affects peptidoglycan synthesis, causing bacterial cell walls to thin, cell membranes to rupture, and the inability to maintain normal morphological and structural integrity, ultimately resulting in bacterial death. Further investigation into the targeting mechanism revealed that, compared to the GlmS inhibitor kanosamaine, GaB can effectively inhibit GlmS enzyme activity ( Figure 3 G), leading to a decrease in glutamate production, and observing bacterial growth curves after incubating GaB with G-6-F, such as Figure 3 H was found to have no effect on bacterial growth, indicating that GaB does not affect the formation of G-6-F and has no significant effect on the enzymes upstream of G-6-F, and indeed acts on GlmS.

[0113] 4. GaB Security Evaluation

[0114] To assess the safety of compound GaB in vivo, we used mouse erythrocytes to detect hemolysis rate, such as... Figure 4 As shown in Figure A, GaB exhibits a very low hemolytic rate in mouse erythrocytes, with negligible hemolysis even at 1024 μg / mL. The cytotoxic IC50 produced by GaB in five mammalian cell lines (MIA PaCa-2, H6C7, AGS, B16, and WPMY-1) was also shown. 50 Values ​​ranged from 3.14 to 9.61 μM. Figure 4B) These results indicate that GaB has a certain degree of safety when administered in vivo. In addition, we conducted a 7-day acute toxicity study in mice, administering three intraperitoneal injections of different concentrations of GaB within 24 hours. We found that mice in the high-dose group (1.6 mg / kg) experienced decreased appetite and gradual weight loss over 7 days, while mice in the low-dose group (0.8 mg / kg) and the positive control group (1.6 mg / kg) showed no abnormal symptoms and their weight increased normally. Figure 4 C); To further characterize the potential of GaB as an antibacterial agent, we also investigated the effects of GaB on organ damage. The results showed ( Figure 4 D) No obvious pathological abnormalities were observed in the liver, kidneys, and heart of the GaB-treated group and the positive control group; however, blood tests on mice revealed that although the high-dose GaB group resulted in blood component levels within the normal range, it caused varying degrees of increase or decrease in white blood cells (WBCs), neutrophils (Gran#), lymphocytes (Lymoh#), hemoglobin (HGB), platelets (PLT), and red blood cells (RBCs). Figure 4 E), showing a certain statistical significance, while the low-dose group and the positive control group showed no significant difference from the blank control group. To further characterize the potential of GaB as an antibacterial drug, we also studied the effect of GaB on organ damage. The results showed ( Figure 4 D) No obvious pathological abnormalities were observed in the liver, kidneys, and heart of the drug group and the positive control group. Based on the above results regarding mouse weight, organ damage, and blood tests, it is indicated that high-dose (1.6 mg / kg) GaB has low toxicity in mice; therefore, we selected a safe dose (0.8 mg / kg) for subsequent experiments.

[0115] 5. GaB is effective in treating infections in the USA300 population.

[0116] Because GaB exhibits strong inhibitory activity against USA300 in vitro, we explored its potential for treating in vivo infections in three animal models of infection. Figure 5 A). Treatment with different concentrations of GaB (5, 10, 15 mg / mL) effectively improved the survival rate of *G. mellonella* larvae infected with USA300. Figure 5 B), which reduced the amount of bacteria in the body ( Figure 5 C) Furthermore, the high-dose GaB (15 mg / mL) showed consistent improvement in survival rate with the positive control group vancomycin (10 mg / mL), achieving a survival rate as high as 80% within 72 hours, indicating comparable efficacy. In addition, we evaluated the efficacy of GaB in two mouse models: a skin wound infection model and a peritonitis-septicemia model. We treated USA300-infected mouse wounds with the drug, observed and measured wound healing, and plotted... Figure 5Figures D and F show that, compared to the blank control group, GaB significantly shortened wound healing time, with effects comparable to vancomycin, indicating that GaB promotes wound tissue repair. Next, the effect of GaB in a mouse peritonitis-septicemia model was tested. After drug treatment, the bacterial load in the liver, heart, spleen, and kidneys of mice was significantly reduced, with effects comparable to vancomycin. Figure 5 E). Results of H&E staining experiments are shown in […]. Figure 5 G. Pathological sections of the infected mouse group showed significant inflammatory cell infiltration in the kidney tissue, tubular dilation, a small amount of deeply stained necrosis of renal tubular epithelial cells, and mild vacuolar degeneration and inflammatory cell infiltration of hepatocytes. Conversely, inflammatory cell infiltration decreased after drug administration. Hepatocytes were distributed around the veins in a regular pattern without obvious abnormalities, and the high-dose GaB group showed comparable efficacy to the positive control group. These findings suggest that GaB has the potential to become an antibiotic.

[0117] In conclusion, aromatic polyketides are promising lead compounds for the development of anti-multidrug resistant bacterial infections.

[0118] The embodiments of this disclosure have been described in detail above with reference to the accompanying drawings. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0119] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0120] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. An aromatic polyketide compound, characterized in that, This includes compound 1, compound 2, compound 3, or compound 4 as shown below:

2. The method for preparing the aromatic polyketide compound according to claim 1, characterized in that, The aromatic polyketide compounds were isolated from the secondary metabolites of the lichen symbiotic Streptomyces sp. QHH-9511.

3. The method for preparing aromatic polyketide compounds according to claim 2, characterized in that, The aromatic polyketide compound was isolated from the ethyl acetate phase of the fermentation broth of Streptomyces sp. QHH-9511.

4. The method for preparing aromatic polyketide compounds according to claim 2 or 3, characterized in that, Specifically, it includes: After fermentation of strain Streptomyces sp. QHH-9511 at 28°C for 8 days in a modified yeast extract liquid medium, the extract was obtained by extraction with ethyl acetate. The extract was separated by normal-phase silica gel column chromatography with dichloromethane-methanol gradient elution under the following conditions: v / v, 100:0 → 100:8; four fractions Fr.1-Fr.4 were obtained; fractions Fr.2 and Fr.3 were separated by reverse-phase RP-C chromatography. 18 The effective components Fr.2.1-Fr.2.2 and Fr.3.1-Fr.3.2 were obtained by elution with MeOH-H2O column at a v / v ratio of 30% to 100%. Then, they were separated and purified by gel column Sephadex LH-20 with MeOH solvent and semi-preparative HPLC at a MeCN-H2O volume concentration of 40%-80% to obtain compounds 1-4.

5. The method for preparing aromatic polyketide compounds according to claim 4, characterized in that, The modified yeast extract liquid culture medium is composed of: 2g peptone, 0.1g FeSO4·7H2O, 0.1g KBr, 4g yeast extract, 10g soluble starch, 1g CaCO3, and 1L water.

6. The use of the aromatic polyketide compound of claim 1 in the preparation of drugs against Gram-positive and / or Gram-negative bacteria.

7. The use of the aromatic polyketide compound of claim 1 in the preparation of drugs against multidrug-resistant bacterial infections.

8. The use of the aromatic polyketide compound of claim 1 in the preparation of drugs for meningitis, intra-abdominal infection and / or skin infection caused by multidrug-resistant bacteria.

9. A drug for treating multidrug-resistant bacterial infections, characterized in that, The drug contains the aromatic polyketide compound of claim 1.

10. An antibiotic, characterized in that, The drug contains the aromatic polyketide compound of claim 1.

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