Application of NSC348884 in preparation of antibacterial drugs

Abstract

The invention discloses an application of NSC348884 in preparation of an antibacterial drug, the bacteria of the NSC348884 are gram-negative bacteria or drug-resistant gram-negative bacteria, and the gram-negative bacteria are acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa or klebsiella pneumoniae; based on an antibacterial drug high-throughput screening technology, it is found from 1280 small molecule compounds that the benzimidazole compound NSC348884 has direct bacteriostasis and sterilization effects on gram-negative bacteria and drug-resistant gram-negative bacteria, and no drug resistance is generated after the benzimidazole compound NSC348884 is continuously applied for 14 days. Further research shows that NSC348884 can interfere with lipid synthesis of gram-negative bacterium cell membranes, inhibit formation of biological membranes and induce lipid metabolism disorder, so that bacteriostatic and bactericidal effects are achieved. In addition, the NSC348884 can also enhance the sensitivity of the drug-resistant bacteria to the imipenem and the polymyxin B.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of NSC348884 in the preparation of antibacterial drugs. Background Technology

[0002] Since the successful development of penicillin in the 1940s, antibiotics and synthetic antimicrobial agents have saved hundreds of millions of lives. However, since the 1990s, only a few structurally optimized antimicrobial agents have been made available, such as daptomycin and tigecycline. Worse still, the widespread use of antimicrobial agents has led to the emergence of many multidrug-resistant bacteria in clinical practice. The continuous emergence of multidrug-resistant bacteria has also significantly limited the use of antibiotics in clinical settings. Currently, carbapenems such as imipenem and meropenem are considered the last line of defense against severe Gram-negative bacterial infections or multidrug-resistant bacteria; however, the emergence of carbapenem-resistant Gram-negative bacteria has led to a continuous decline in the sensitivity of carbapenems.

[0003] In 2017, the World Health Organization (WHO) released its first Priority Bacterial Pathogens List (BPPL), categorizing antibiotic-resistant bacteria into three priority levels—critical, high, and medium—based on their threat to human health. Critical priority bacteria include carbapenem-resistant Acinetobacter baumannii (CRAB), carbapenem-resistant Pseudomonas aeruginosa (CRPA), and carbapenem-resistant Enterobacteriaceae that produce extended-spectrum β-lactamases (ESBLs), primarily carbapenem-resistant Escherichia coli (CREC) and carbapenem-resistant Klebsiella pneumoniae (CRKP).

[0004] In 2024, the resistance rates of Acinetobacter baumannii isolated clinically in my country to carbapenem antibiotics such as imipenem and meropenem reached as high as 64.5% and 64.7%, respectively, leaving infected patients facing a critical situation where no drugs were available. The development of antibacterial drugs targeting severe Gram-negative bacterial infections or multidrug-resistant bacteria is urgently needed.

[0005] The combined use of sensitizers to enhance the sensitivity of drug-resistant bacteria to antibiotics or antimicrobial drugs is another effective strategy in the development of antibiotics or antimicrobial drugs, such as imipenem / cilastatin / ralobacterium (Recarbrio) and imipenem / formobacterium. Among them, ralobacterium and formobacterium, as β-lactamase inhibitors, enhance the sensitivity of imipenem.

[0006] NSC348884 is an inhibitor of nucleophosmin (NPM), CAS number 81624-55-7. Current research indicates that NSC348884 can disrupt oligomer formation, induce apoptosis, and inhibit the proliferation of various cancer cells. 50 The range is 1.7-4.0 μM. [1] Furthermore, NSC348884 possesses repair capabilities following myocardial infarction. [2] However, there are no reports on its application in antibacterial activity and enhancing the sensitivity of antibacterial drugs.

[0007] References:

[0008] 1.Qi W,Shakalya K,Stejskal A,Goldman A,Beeck S,Cooke L,MahadevanD.NSC348884, anucleophosmin inhibitor disrupts oligomer formation and inducesapoptosis in human cancer cells.Oncogene.2008,27(30):4210-20.

[0009] 2. Zhang S, Zhang Y, Duan X, Wang B, Zhan Z. Targeting NPM1 EpigeneticallyPromotes Postinfarction Cardiac Repair by Reprogramming Reparative MacrophageMetabolism.Circulation.2024,149(25):1982-2001. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of NSC348884 in the preparation of antibacterial drugs.

[0011] A second objective of this invention is to provide the use of NSC348884 in the preparation of antibiotic sensitizing drugs.

[0012] The technical solution of this invention is summarized as follows:

[0013] Application of NSC348884 in the preparation of antibacterial drugs.

[0014] The bacteria are Gram-negative bacteria or drug-resistant Gram-negative bacteria.

[0015] The Gram-negative bacteria are Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, or Klebsiella pneumoniae.

[0016] The drug is one or more of aminoglycoside antibiotics, β-lactam antibiotics, quinolone antibacterial drugs, and sulfonamide antibacterial drugs.

[0017] Aminoglycoside antibiotics include: amikacin, gentamicin, and tobramycin;

[0018] β-lactam antibiotics include: meropenem, imipenem, cefoperazone / sulbactam, ampicillin / sulbactam, ceftriaxone, ceftazidime, cefepime, piperacillin / tazobactam, and piperacillin;

[0019] Quinolone antibiotics include: ciprofloxacin and levofloxacin;

[0020] Sulfonamide antibiotics include: compound sulfamethoxazole.

[0021] Application of NSC348884 in the preparation of antibiotic sensitizers.

[0022] The antibiotic is imipenem or polymyxin B.

[0023] Advantages of this invention:

[0024] This invention, based on high-throughput screening technology for antimicrobial drugs, discovered that the benzimidazole compound NSC348884 exhibits direct antibacterial and bactericidal effects against Gram-negative bacteria and drug-resistant Gram-negative bacteria from 1280 small molecule compounds, with no drug resistance developing after 14 days of continuous application. Further research showed that NSC348884 interferes with lipid synthesis in the cell membrane of Gram-negative bacteria, inhibits biofilm formation, and induces lipid metabolism disorders, thereby exerting its antibacterial and bactericidal effects. In addition, NSC348884 can enhance the sensitivity of drug-resistant bacteria to imipenem and polymyxin. Attached Figure Description

[0025] Figure 1 The drug resistance spectrum of 14 clinically isolated Gram-negative bacteria.

[0026] Figure 2 The minimum bactericidal concentration (MBC) of NSC348884 against standard strain ATCC 19606 and drug-resistant Acinetobacter baumannii CRAB6-9 is shown in the figure.

[0027] Figure 3 This is a graph showing the evolution of resistance to NSC348884 in the standard strain ATCC 19606 and drug-resistant Acinetobacter baumannii CRAB6-9 during 14 consecutive days of drug administration.

[0028] Figure 4The image shows the therapeutic effect of NSC348884 on mice infected with drug-resistant Acinetobacter baumannii.

[0029] Figure 5 The diagram shows the effects of NSC348884 on disrupting the cell membranes of standard strain ATCC 19606 and drug-resistant Acinetobacter baumannii CRAB6-9, interfering with biomembrane synthesis, and inducing lipid metabolism disorders.

[0030] Figure 6 The graph shows the inhibitory and sensitizing effects of NSC348884 in combination with imipenem or polymyxin B on drug-resistant Gram-negative bacilli. Detailed Implementation

[0031] The present invention will now be clearly and completely described in conjunction with the embodiments and 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.

[0032] Unless otherwise specified, the experimental methods and breakpoint interpretations in the following examples are based on the Clinical and Laboratory Standards Institute (CLSI) standards (CLSIM100-Ed35).

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. NSC348884, phosphatidylcholine, cholesterol, DSPE-MPEG2000, imipenem, polymyxin B, and cyclophosphamide were purchased from Shanghai Maclean Biotechnology Co., Ltd. MH broth and LB medium were purchased from Beijing Solarbio Science & Technology Co., Ltd., and AG RNAex Pro RNA extraction reagent was purchased from Hunan Aikerui Biotechnology Co., Ltd.

[0034] The strain information in the following examples is as follows:

[0035] The standard strains of Acinetobacter baumannii (ATCC 19606), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Klebsiella pneumoniae (ATCC 700603) were all purchased from [unclear - likely a company name]. Company. (April 2023, USA, https: / / www.microbiologics.com)

[0036] Drug-resistant Acinetobacter baumannii CRAB1-2, CRAB1-4, CRAB2-3, CRAB2-6, CRAB4-2, CRAB5-5, CRAB5-7, CRAB5-8, CRAB6-8, CRAB6-9, CRAB6-10, CREC3-8, CRPA6-10, and CRKP3-3 were all clinically isolated drug-resistant strains from the Department of Laboratory Medicine, Second Affiliated Hospital of Dalian Medical University. The isolation and identification procedures were guided by the "Basic Technical Standards for Clinical Microbiology Laboratory Testing" (WS / T 805–2022).

[0037] Example 1

[0038] Inhibitory effects of NSC348884 on Gram-negative bacteria and drug-resistant Gram-negative bacteria

[0039] Fourteen clinically isolated drug-resistant Gram-negative bacteria, CRAB1-2, CRAB1-4, CRAB2-3, CRAB2-6, CRAB4-2, CRAB5-5, CRAB5-7, CRAB5-8, CRAB6-8, CRAB6-9, CRAB6-10, CREC3-8, CRPA6-10, and CRKP3-3, were first identified using the bioMérieux VITEK2 compact fully automated bacterial identification and antimicrobial susceptibility analyzer. Antimicrobial susceptibility testing was also performed on 16 commonly used clinical antimicrobial drugs / combination preparations to construct drug resistance profiles.

[0040] The results are as follows Figure 1 As shown, 13 clinical isolates of Gram-negative bacteria (including 11 CRAB, 1 CREC, and 1 CRKP) were sensitive to tigecycline but exhibited extensive resistance to 15 other clinical drugs / combination formulations (cefotaxime / sulbactam, amikacin, ampicillin / sulbactam, ciprofloxacin, trimethoprim-sulfamethoxazole, meropenem, gentamicin, ceftriaxone, ceftazidime, cefepime, tobramycin, imipenem, levofloxacin, piperacillin, and piperacillin / tazobactam). The clinical isolate CRPA6-10 showed resistance to six clinical drugs / combination formulations, including imipenem, meropenem, tigecycline, trimethoprim-sulfamethoxazole, ampicillin / sulbactam, and ceftriaxone, but was sensitive to ten clinical drugs / combination formulations, including cefoperazone / sulbactam, amikacin, ciprofloxacin, gentamicin, ceftazidime, cefepime, tobramycin, levofloxacin, piperacillin, and piperacillin / tazobactam.

[0041] Experimental bacterial strains ATCC 19606, ATCC 25922, ATCC 27853, ATCC 700603, CRAB1-2, CRAB1-4, CRAB2-3, CRAB2-6, CRAB4-2, CRAB5-5, CRAB5-7, CRAB5-8, CRAB6-8, CRAB6-9, CRAB6-10, CREC3-8, CRPA6-10, and CRKP3-3 were inoculated into MH broth and cultured overnight at 37°C with shaking until the logarithmic growth phase. 2 mL of each bacterial culture was centrifuged at 5000 × g for 5 min at room temperature, the bacterial pellet was resuspended in sterile physiological saline, and the bacterial concentration was adjusted to 0.5 McFarland turbidity (approximately 1.5 × 10⁻⁶). 8 (CFU / mL). Subsequently, each bacterial suspension was diluted 100-fold with MH broth, and 100 μL of the bacterial suspension was inoculated into a 96-well plate. NSC348884 was dissolved in DMSO to prepare a 6.4 mg / mL solution. Using a two-fold dilution method, NSC348884 was diluted with MH broth to concentrations of 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0 μg / mL. 100 μL of MH broth containing different concentrations of NSC348884 was added to each of the above-mentioned 96-well plates containing the bacterial suspension, and the mixture was pipetted to achieve final drug concentrations of 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0 μg / mL (i.e., control group). Each sample group was set up in triplicate and incubated at 37℃ for 20 h. The OD of each well was measured using a microplate reader. 600 Value, calculate inhibition rate (%) = [(OD value)] 对照组 -OD 各孔 ) / OD 对照组 The lowest drug concentration with an inhibition rate of ≥90% is considered the minimum inhibitory concentration (MIC).

[0042] The results are shown in Table 1. NSC348884 exhibited good antibacterial activity against Gram-negative bacteria and drug-resistant Gram-negative bacteria (Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae), with a MIC range of 2-64 μg / mL. Among them, Acinetobacter baumannii was the most sensitive to NSC348884, with a MIC of 2-8 μg / mL.

[0043] Table 1. Minimum Inhibitory Concentration (MIC) of NSC348884 against Gram-negative and Drug-Resistant Gram-Negative Bacteria

[0044]

[0045] Example 2

[0046] Evaluation of the bactericidal and resistance-inducing effects of NSC348884 on Gram-negative bacteria and drug-resistant Gram-negative bacteria

[0047] The following experimental studies were conducted using Acinetobacter baumannii (ATCC 19606 and clinically isolated drug-resistant strain CRAB6-9) as examples. The above strains were inoculated into MH broth and cultured at 37°C with shaking until the logarithmic growth phase. 2 mL of each bacterial suspension was centrifuged at 5000×g for 5 min at room temperature. The bacterial pellet was resuspended in sterile physiological saline, and the bacterial concentration was adjusted to 0.5 McFarland turbidity (approximately 1.5 × 10⁻⁶). 8 (CFU / mL). Subsequently, the bacterial suspension was diluted 100-fold with MH broth, and 100 μL of the suspension was inoculated into a 96-well plate. NSC348884 was dissolved in DMSO to prepare a 6.4 mg / mL solution. Using a two-fold dilution method, NSC348884 was diluted with MH broth to concentrations of 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0 μg / mL. 100 μL of MH broth containing different concentrations of NSC348884 was added to each of the above-mentioned 96-well plates containing the bacterial suspension, and the mixture was pipetted to achieve final drug concentrations of 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0 μg / mL (i.e., control groups). The blank control group contained only culture medium and no bacterial cells; the drug control group contained only NSC348884 and culture medium, without bacterial cells. Each sample group was divided into 3 parallel groups and incubated in a 37℃ incubator for 20 hours.

[0048] To clarify whether NSC348884 has a direct bactericidal effect against Gram-negative bacteria and drug-resistant Gram-negative bacteria, after the above-mentioned 96-well plates were incubated, 50 μL of bacterial culture from each well was aspirated and spread onto LB agar plates. After the liquid on the plates dried, they were inverted and placed back in an incubator at 37°C for overnight incubation. The next day, the number of colonies visible on the solid medium was recorded. The lowest drug concentration at which the colony count decreased by 99.9% or no colony growth occurred was defined as the minimum bactericidal concentration (MBC). The results are as follows: Figure 2 As shown, NSC348884 had MBC values ​​of 8 μg / mL and 16 μg / mL against ATCC 19606 and CRAB6-9, respectively, which are 2-4 times the MIC values, indicating that NSC348884 has bactericidal activity against ATCC 19606 and CRAB6-9.

[0049] To study the drug resistance effects of NSC348884 on Gram-negative bacteria and drug-resistant Gram-negative bacteria, the OD values ​​of each well were measured using a microplate reader. 600 Value, calculate inhibition rate (%) = [(OD value)] 对照组 -OD 各孔 ) / OD 对照组The lowest drug concentration with an inhibition rate ≥90% (×100%) is considered the minimum inhibitory concentration (MIC), which is the first generation. Bacteria in 96-well plates with a concentration of 0.5×MIC are cultured to the exponential phase, then inoculated into 96-well plates for a second NSC348884 susceptibility test. This process is repeated for 14 consecutive generations to detect the evolution trend of bacterial resistance to NSC348884. Results are as follows... Figure 3 As shown, after 14 days of continuous induction, the MIC of ATCC 19606 remained unchanged, while the MIC of CRAB6-9 fluctuated within a 1-2 fold range, with no significant drug resistance observed. These results indicate that continuous use for 14 days does not affect the susceptibility of Gram-negative bacteria and drug-resistant Gram-negative bacteria to NSC348884.

[0050] Example 3

[0051] The therapeutic effect of NSC348884 on mice infected with drug-resistant Gram-negative bacteria

[0052] We conducted relevant experimental studies using drug-resistant Acinetobacter baumannii (clinically isolated drug-resistant strain CRAB6-9) as an example.

[0053] 1) Preparation of NSC348884 liposomes. Weigh out 6 mg of NSC348884; 81 mg of phosphatidylcholine; 9 mg of cholesterol; and 6 mg of DSPE-MPEG2000 (commercial). Dissolve the weighed NSC348884, phosphatidylcholine, cholesterol, and DSPE-MPEG2000 in chloroform to form an organic phase. Remove the chloroform using a rotary evaporator at 50–55 °C, forming a uniform thin film on the flask wall. Add 5 mL of physiological saline to the rotary evaporator and hydrate by stirring at 50–55 °C for 1 h. Place the hydrated liposome solution in an ultrasonic disruptor and sonicate for 5 min at 100 W under ice bath conditions to reduce particle size, improve uniformity, and promote the formation of monolayer liposomes. The liposome solution prepared by ultrasound was centrifuged at 10,000 rpm for 5 min. The liposome supernatant was filtered through a 0.22 μm PES filter membrane to remove large particles and microorganisms, and a clear and homogeneous liposome preparation was obtained for subsequent experiments.

[0054] Preparation of blank liposomes: NSC348884 was not added in the preparation of NSC348884 liposomes as described above.

[0055] 2) Construction and treatment of bacteremia model in mice infected with drug-resistant Acinetobacter baumannii. Eighteen 6-8 week old male C57BL / 6N mice (approximately 20-22g) were randomly divided into 3 groups of 6 mice each.

[0056] Four days prior to the experiment, all mice were intraperitoneally injected with cyclophosphamide 100 mg / kg for three consecutive days to induce a decrease in their immunity. The mice were fasted for 8 hours before the experiment. On the day of the experiment, the mice were first anesthetized with tribromoethanol, their limbs were fixed with medical tape, and the left jugular vein was carefully separated from the muscle tissue. 0.1 mL of CRAB6-9 bacterial suspension (10...) was then collected. 7 A bacteremia model was constructed by slowly injecting CFU into the jugular vein.

[0057] Ten minutes later, the drug administration began. The model group was given an equal volume of blank liposomes (solvent: physiological saline); the imipenem positive control group (hereinafter referred to as the imipenem group) was given a dose of 12.5 mg / kg (solvent: physiological saline); and the NSC348884 liposome treatment group (hereinafter referred to as the NSC348884 group) was given a dose of 12.5 mg / kg (solvent: physiological saline).

[0058] According to different groups, drugs were slowly administered via intravenous drip using a medical micro-infusion pump at a flow rate of 0.5 mL / h. Six hours after model establishment, the mice's heads were wiped with alcohol-soaked cotton balls to disinfect the surface bacteria, and blood was collected by enucleation of the eyeballs. The mice were then euthanized by cervical dragging, and the carcasses were immersed in 75% ethanol for surface sterilization. Lung tissue was harvested, washed three times with physiological saline, and a portion of the tissue was immersed in 10% formalin, while the remaining tissue was frozen at -80°C.

[0059] 3) Detection of bacterial load in blood. Perform a 10-fold serial dilution of the blood (using physiological saline as the solvent): 10 0 10 1 10 2 10 3 For each dilution, take 10 μL of sample and drop it onto the corresponding plate (the plate is divided into 2 rows and 4 columns, i.e., one column for each dilution, and the technique is repeated twice). After drying, invert the plate and incubate overnight at 37°C. Select colonies with a number of 10-30 for counting. The bacterial count calculation formula is: log... 10 (bacterial count / mL blood) = log 10 (Number of bacteria × 100 × dilution factor).

[0060] 4) Detection of inflammatory factors in lung tissue. The expression levels of inflammatory factors in lung tissue were detected using qPCR. The steps were as follows: Weigh 25-30 mg of lung tissue into a 1.5 mL RNase-free tube, add 500 μL of AG RNAex Pro RNA extraction reagent, homogenize the tissue, and let it stand at room temperature for 5 min. Transfer the supernatant to another sterile RNase-free tube. Add 100 μL of chloroform, vortex vigorously until emulsified, let stand for 5 min, and centrifuge at 12,000 × g, 4 °C for 5 min. Take 200 μL of the uppermost aqueous phase into a new EP tube, add 250 μL of pre-chilled isopropanol, invert and mix well, let stand at room temperature for 10 min, and centrifuge at 12,000 × g, 4 °C for 10 min. Discard the supernatant, add 500 μL of pre-chilled ethanol (80%) to wash the RNA precipitate, invert and mix well, and centrifuge at 7500 × g, 4 °C for 5 min. Open the centrifuge tube cap, discard the supernatant, and dry at room temperature for 5 min. Dissolve the precipitate in sterile, enzyme-free water. Measure the concentration of the dissolved RNA and then reverse transcribe it into cDNA for later use. Both upstream and downstream primer sequences (including the internal reference gene β-actin) were purchased from Beijing Liuhe BGI Genomics Co., Ltd. qPCR was performed according to the following reaction system: 10 μL 2×SYBR Mix; 0.4 μL upstream primer; 0.4 μL downstream primer; 1 μL cDNA template (100 ng); finally, add sterile, enzyme-free water to a final volume of 20 μL. Data were normalized using the internal reference gene β-actin and 2... -ΔΔCT Quantitative analysis of the mRNA expression levels of inflammatory factors.

[0061] 5) Detection of lung tissue pathological damage. Partial lung tissue from mice was fixed in 10% formalin solution for 24 hours. After paraffin embedding and sectioning, the paraffin tissue sections were stained with hematoxylin and eosin (H&E) and observed and photographed under a microscope.

[0062] The results are as follows Figure 4 As shown in the figure, imipenem treatment resulted in only a slight decrease in blood bacterial load in mice, with no statistically significant difference compared to the model group. NSC348884 liposome treatment significantly inhibited the in vivo proliferation of drug-resistant Acinetobacter baumannii CRAB6-9, reducing blood bacterial load by 97.9% compared to the model group. Lung tissue inflammatory factor detection revealed a significant decrease in the mRNA expression levels of TNF-α and IL-6 in lung tissue after NSC348884 liposome treatment. Simultaneously, H&E staining results indicated that NSC348884 liposome treatment resulted in well-defined alveolar tissue structure, reduced inflammatory symptoms, and alleviated symptoms such as alveolar epithelial cell lesions or necrosis, while maintaining alveolar morphology. These results suggest that NSC348884 liposomes can effectively reduce the bacterial load of drug-resistant Acinetobacter baumannii-infected mice and alleviate lung inflammatory damage.

[0063] Example 4

[0064] NSC348884 disrupts the cell membranes of Gram-negative and drug-resistant Gram-negative bacteria, interferes with biomembrane synthesis, and induces lipid metabolism disorders.

[0065] We conducted relevant experimental studies using Acinetobacter baumannii (ATCC 19606 and clinically isolated drug-resistant strain CRAB6-9) as examples.

[0066] 1) Scanning electron microscopy observation of the effect of NSC348884 on cell morphology. Experimental strains (Acinetobacter baumannii standard strain ATCC 19606 and clinically isolated drug-resistant strain CRAB6-9) were inoculated into LB broth and cultured at 37°C with shaking until the logarithmic growth phase. Each strain was divided into two groups: 0 μg / mL NSC348884 and 8 μg / mL NSC348884, with three replicates per group. The logarithmic growth phase bacterial suspensions were diluted 1:100 and inoculated into fresh LB broth according to the above groups, and cultured at 37°C and 200 rpm for 2 h with shaking. After culture, DMSO or NSC348884 was added to each group, and the cells were returned to 37°C for another 2 h with shaking. Cells were centrifuged at 5000×g for 5 min to pellet the cells, and washed three times with PBS to thoroughly remove residual culture medium. The cell pellet was resuspended in 2.5% glutaraldehyde fixative and fixed overnight at 4°C. Centrifuge at 5000×g for 2 min to remove fixative, and wash twice with PBS. Then, fix the sample with 1% osmium tetroxide aqueous solution at room temperature for 1 h. Dehydrate stepwise with ethanol gradients, replacing ethanol with isoamyl acetate twice. Dry and then deposit a gold film. Finally, observe the ultrastructural changes of the bacteria using a Hitachi SU8100 scanning electron microscope.

[0067] 2) NSC348884 inhibits biofilm formation and promotes the degradation of mature biofilms. The experimental strains (Acinetobacter baumannii standard strain ATCC 19606 and clinically isolated drug-resistant strain CRAB6-9) were inoculated into MH broth and cultured overnight at 37°C with shaking until the logarithmic growth phase. 2 mL of each culture was centrifuged at 5000×g for 5 min at room temperature, and the bacterial pellet was resuspended in sterile physiological saline. The bacterial concentration was adjusted to 0.5 McFarland turbidity (approximately 1.5 × 10⁻⁶). 8(CFU / mL). Subsequently, the bacterial suspension was diluted 100-fold with MH broth, and 100 μL of the bacterial suspension was inoculated into a 96-well plate. The biofilm inhibition experiment was conducted as follows: Polymyxin B was used as a positive control drug. Four groups of NSC348884 and polymyxin B were set up according to concentration gradients: Control (0 μg / mL), 0.25×MIC, 0.5×MIC, and MIC. Following these groupings, 100 μL of NSC348884 and polymyxin B dissolved in MH broth (at different concentrations such as 0 μg / mL, 0.5×MIC, MIC, and 2×MIC) were added to the bacterial suspension in the 96-well plate and mixed thoroughly. The final concentrations of NSC348884 and polymyxin B were 0 μg / mL, 0.25×MIC, 0.5×MIC, and MIC, respectively. The plates were incubated at 37°C for 24 h to allow the bacteria to form a biofilm that adhered to the bottom of the wells. Carefully aspirate and discard the supernatant. Wash twice with PBS, then add 100 μL of methanol to fix the biofilm adhering to the well plate for 30 min. Aspirate the methanol and stain the biofilm with 0.1% (w / v) crystal violet solution for 15 min. Aspirate the crystal violet solution and wash with pure water. Add 100 μL of anhydrous ethanol to each well, repeatedly pipetting to ensure the biofilm is evenly dissolved in the anhydrous ethanol, and shake on a shaker at room temperature for 30 min. Measure the OD of the biofilm after crystal violet staining using a microplate reader. 590 The final biofilm formation amount was standardized as a percentage of the Control group (% of Control). The steps to promote the decomposition of mature biofilms are as follows: Adjust the above to 0.5 McFarland turbidity (approximately 1.5 × 10⁻⁶). 8 Bacterial suspension (CFU / mL) was diluted 100-fold with MH broth. 100 μL of the suspension was inoculated into a 96-well plate and incubated at 37°C for 24 h to form a mature biofilm. The plate was then washed three times with PBS to remove free bacteria before use. Four concentration gradients were established for NSC348884 and polymyxin B: Control (0 μg / mL), MIC, 2×MIC, and 4×MIC. Following these groupings, 100 μL of NSC348884 and polymyxin B dissolved in MH broth (at different concentrations: 0 μg / mL, MIC, 2×MIC, and 4×MIC) were added to each 96-well plate and incubated at 37°C for 24 h. Subsequent biofilm detection using crystal violet staining followed the same method. The final biofilm residue was standardized as a percentage of the Control group (% of control).

[0068] 3) Exogenous addition of oleic acid conferred resistance to NSC348884 in Acinetobacter baumannii. Experimental strains (Acinetobacter baumannii standard strain ATCC 19606 and clinically isolated drug-resistant strain CRAB6-9) were inoculated into MH broth and cultured overnight at 37°C with shaking until the logarithmic growth phase. 2 mL of each bacterial suspension was centrifuged at 5000×g for 5 min at room temperature, the bacterial pellet was resuspended in sterile physiological saline, and the bacterial concentration was adjusted to 0.5 McFarland turbidity (approximately 1.5 × 10⁻⁶). 8 (CFU / mL). Subsequently, the bacterial culture was diluted 100-fold with MH broth (or MH broth containing 40 μg / mL oleic acid), and 100 μL of the bacterial culture was inoculated into a 96-well plate. NSC348884 was dissolved in DMSO to prepare a 6.4 mg / mL solution, and then diluted with MH broth to 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0 μg / mL using the two-fold dilution method. 100 μL of MH broth containing different concentrations of NSC348884 was added to the corresponding 96-well plates containing bacterial suspension (or bacterial suspension containing 40 μg / mL oleic acid). The mixture was pipetted and stirred to achieve final NSC348884 concentrations of 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0 μg / mL, and a final oleic acid concentration of 20 μg / mL. Three replicates were prepared for each concentration, and the plates were incubated at 37°C for 20 h. The OD values ​​of each well were measured using a microplate reader. 600 Value, based on the endpoint OD 600 As a result, a line graph was plotted, and the effects of different drug treatments on the growth of the strain were compared to investigate the effect of oleic acid on the antibacterial activity of NSC348884.

[0069] The results are as follows Figure 5 As shown, the membrane surfaces of ATCC 19606 and CRAB6-9 strains treated with NSC348884 (8 μg / mL) exhibited shrinkage and collapse, with some cells clearly rupturing, strongly indicating that NSC348884 damaged the bacterial membrane. Similar to polymyxin B, NSC348884 significantly inhibited biofilm formation and promoted the decomposition of mature biofilms in a concentration-dependent manner, demonstrating a strong ability to attack biofilms and contributing to enhanced bactericidal effects. Metabolomics analysis revealed lipid metabolism disorders; exogenous addition of oleic acid enhanced resistance to NSC348884-induced membrane-targeting stress, improving strain tolerance.

[0070] Example 5

[0071] Application of NSC348884 in the preparation of antibiotic sensitizers

[0072] The effect of NSC348884 on the antibacterial activity of imipenem or polymyxin B was determined using the checkerboard method. Experimental strains CRAB6-9, CREC3-8, CRPA6-10, and CRKP3-3 were inoculated into MH broth and cultured overnight at 37°C with shaking until the logarithmic growth phase. 2 mL of each bacterial suspension was centrifuged at 5000 × g for 5 min at room temperature, the bacterial pellet was resuspended in sterile physiological saline, and the bacterial concentration was adjusted to 0.5 McFarland turbidity (approximately 1.5 × 10⁻⁶). 8 (CFU / mL). Subsequently, the bacterial culture was diluted 100 times with MH broth for later use. Preparation of MH broth drug solutions: Imipenem (0, 1, 2, 4, 8, 16, 32, 64 μg / mL) or polymyxin B (0, 0.125, 0.25, 0.5, 1, 2, 4, 8 μg / mL) dissolved in MH broth was added sequentially from left to right in each column as a concentration gradient. 200 μL was added to the first well, and 100 μL to the remaining wells. Then, 2 μL (1.6 mg / mL) was added to the first well. Prepare a stock solution of NSC348884 (6.4 mg / mL) in DMSO. After mixing, use an 8-channel pipette to add 100 μL of the mixture to the next row. Dilute the solution by a factor of two and mix thoroughly. Finally, discard the 100 μL of liquid. The concentrations of NSC348884 in rows 1 to 8 are 16, 8, 4, 2, 1, 0.5, 0.25, 0 μg / mL or 64, 32, 16, 8, 4, 2, 1, 0 μg / mL, respectively. Continue adding 100 μL of the prepared MH broth bacterial culture to the 96-well plate and mix well. The final concentrations of imipenem are 0, 0.5, 1, 2, 4, 8, 16, and 32 μg / mL; the final concentrations of polymyxin B are 0, 0.0625, 0.125, 0.25, 0.5, 1, 2, and 4 μg / mL; and the final concentrations of NSC348884 are 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0 μg / mL or 32, 16, 8, 4, 2, 1, 0.5, and 0 μg / mL. Incubate at 37°C for 20 h and record the OD using a microplate reader. 600 The control group consisted of 0 μg / mL bacterial culture containing no drug. The inhibition rate (%) was calculated as follows: = [(OD2000) / [0.05] ... 对照组 -OD 各孔 ) / OD 对照组 The lowest drug concentration with an inhibition rate ≥90% is considered the minimum inhibitory concentration (MIC). The combined inhibitory index (FICI) is calculated using the following formula: FICI = (MIC × 100%) A (United) / (MIC) A (single) + (MIC) B (United) / (MIC) BIndividually), A: imipenem or polymyxin B; B: NSC348884. In this invention, the minimum FICI is taken as the final basis for judging the combined effect, and the criteria for judging the synergistic antibacterial effect are as follows: FICI ≤ 0.5: synergistic effect; 0.5 < FICI ≤ 1.0: additive effect; 1.0 < FICI ≤ 4.0: no effect; FICI > 4.0: antagonistic effect.

[0073] The results are as shown Figure 6 below. Calculated according to the above FICI formula, Figure 6 the red pentagrams in it are the wells where the minimum FICI is located. The combination of NSC348884 and imipenem showed an additive effect against CRAB6-9 (FICI = 0.75), CREC3-8 (FICI = 0.56), and CRPA6-10 (FICI = 0.75), and a synergistic effect against CRKP3-3 (FICI = 0.50). The combination of NSC348884 and polymyxin B showed an additive effect against CRAB6-9 (FICI = 0.75) and CRKP3-3 (FICI = 0.53), and a synergistic effect against CREC3-8 (FICI = 0.25) and CRPA6-10 (FICI = 0.50). These indicate that NSC348884 can enhance the antibacterial activity of imipenem or polymyxin B against drug-resistant Gram-negative bacteria, has a broad-spectrum synergistic and sensitizing effect, and can be further used for the development of antibiotic-sensitizing drugs.

Claims

1. Application of NSC348884 in the preparation of antibacterial drugs.

2. The application according to claim 1, characterized in that: The bacteria are Gram-negative bacteria or drug-resistant Gram-negative bacteria.

3. The application according to claim 2, characterized in that: The Gram-negative bacteria are Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, or Klebsiella pneumoniae.

4. The application according to claim 2, characterized in that: The drug is one or more of aminoglycoside antibiotics, β-lactam antibiotics, quinolone antibacterial drugs, and sulfonamide antibacterial drugs.

5. Application of NSC348884 in the preparation of antibiotic sensitizers.

6. The application according to claim 5, characterized in that: The antibiotic is imipenem or polymyxin B.

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