Use of bms-833923 and derivatives thereof and medicaments

Abstract

The application discloses application of BMS-833923 and derivatives thereof and a medicine, and belongs to the technical field of antibiotics. The BMS-833923 and the derivatives thereof can be used as colistin adjuvants to jointly inhibit or eliminate gram-negative bacteria, and can also independently inhibit or eliminate gram-positive bacteria. The gram-negative bacteria can include at least one of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, pan-drug-resistant strains BAA-1800, BAA-1794 and BAA-1792, and the gram-positive bacteria can include at least one of Staphylococcus aureus and Bacillus subtilis. By taking the BMS-833923 and the derivatives thereof as adjuvants of colistin, the therapeutic index of the colistin can be effectively expanded, and lower, non-toxic doses of the colistin can be used in clinical treatment of effective treatment of drug-resistant bacterial infections.

Description

Technical Field

[0001] This invention relates to the field of antibiotic technology, and more specifically, to the application and pharmaceutical use of BMS-833923 and its derivatives. Background Technology

[0002] The increasing resistance of many common bacterial pathogens to antibiotics has become a global crisis threatening human life. "ESKAPE" pathogens—Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli—represent a major challenge among these multidrug-resistant pathogens.

[0003] Currently, bacteria have developed resistance to almost all clinically available antibiotics, and in many cases, there is a lack of effective means to control and treat bacterial infections. Therefore, there is an urgent need to develop new antibiotics to alleviate this crisis. However, in recent years, the discovery of new antibiotics has become increasingly difficult, and fewer and fewer new antibiotics are under development. Some older antibiotics, abandoned due to their toxic side effects, have had to be reintroduced into clinical practice as a means of treating infections.

[0004] Polymyxins are polycationic peptide antibiotics developed in the 1940s. These antibiotics kill bacteria by disrupting their cell membranes. The cationic charge in polymyxins electrostatically binds to the negatively charged lipopolysaccharides on the outer membrane of Gram-negative bacteria, then displaces calcium and magnesium ions that stabilize the cell membrane, ultimately disrupting its integrity. Although polymyxins (such as colistin) are effective at killing bacteria, they were previously abandoned in clinical use due to their significant nephrotoxicity and neurotoxicity. Recently, the lack of effective treatments for multidrug-resistant bacteria (such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae) has led to high morbidity and mortality rates among patients infected with these bacteria. As an effective treatment, colistin has been reintroduced into clinical practice in recent years to treat superbug infections, especially Gram-negative superbug infections, and is often used as a last resort. However, studies have shown that an average of 30% of patients receiving colistin treatment experience at least mild nephrotoxicity.

[0005] Therefore, there is an urgent need for a method to expand the therapeutic index of colistin so that it can be used clinically at lower, non-toxic doses, thereby reducing its toxic side effects.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] One of the objectives of this invention is to provide an application of BMS-833923 and its derivatives as colistin adjuvants, such as for co-treatment with colistin against drug-resistant bacteria, which can effectively expand the therapeutic index of colistin and allow for the clinical use of lower, non-toxic doses of colistin for the effective treatment of drug-resistant bacterial infections.

[0008] The second objective of this invention is to provide an antibacterial drug whose active ingredients include BMS-833923 and its derivatives and colistin, which can reduce the concentration of antibiotics with toxic side effects used in clinical practice by enhancing the bactericidal activity of antibiotics (colistin) and avoid their side effects on patients.

[0009] This application can be implemented as follows:

[0010] In one aspect, this application provides an application of BMS-833923 and its derivatives, specifically, BMS-833923 and its derivatives are used as colistin adjuvants.

[0011] Secondly, this application provides for the application of BMS-833923 and its derivatives in combination with colistin for the treatment of bacteria.

[0012] In an optional implementation, the bacteria are Gram-negative bacteria.

[0013] In an optional implementation, the Gram-negative bacteria are in the logarithmic growth phase.

[0014] In an optional implementation, 1×MIC 50 Add 5-15 μg / mL of BMS-833923 and its derivatives to colistin-treated Gram-negative bacteria.

[0015] In an optional implementation, 1×MIC 50 Add 10 μg / mL of BMS-833923 and its derivatives to colistin-treated Gram-negative bacteria.

[0016] In an optional implementation, BMS-833923 and its derivatives are used in combination with colistin to inhibit or eliminate Escherichia coli.

[0017] In an optional implementation, BMS-833923 and its derivatives are used in combination with colistin to inhibit or eliminate Klebsiella pneumoniae.

[0018] In an optional implementation, BMS-833923 and its derivatives are used in combination with colistin to inhibit or eliminate Acinetobacter baumannii.

[0019] In an optional implementation, BMS-833923 and its derivatives are used in combination with colistin to inhibit or eliminate Pseudomonas aeruginosa.

[0020] In an optional embodiment, BMS-833923 and its derivatives are used in combination with colistin to inhibit or eliminate at least one of the pan-drug-resistant strains BAA-1800, BAA-1794 and BAA-1792.

[0021] Thirdly, this application provides for the use of BMS-833923 and its derivatives for the independent inhibition or elimination of Gram-positive bacteria.

[0022] In an optional embodiment, the Gram-positive bacteria include at least one of Staphylococcus aureus and Bacillus subtilis.

[0023] Fourthly, this application provides an antibacterial drug whose active ingredients include BMS-833923 and its derivatives and colistin.

[0024] The antibacterial drug applied for is a drug that inhibits or eliminates at least one of the following: Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, and pan-drug-resistant strains BAA-1800, BAA-1794, and BAA-1792.

[0025] The beneficial effects of this application include:

[0026] By using BMS-833923 and its derivatives as adjuvants to colistin, and co-treating it with colistin against drug-resistant bacteria, the therapeutic index of colistin can be effectively expanded, allowing for the clinical use of lower, non-toxic doses of colistin for the effective treatment of drug-resistant bacterial infections. The active ingredient, containing both BMS-833923 and its derivatives and the antibacterial properties of colistin, can enhance the bactericidal activity of antibiotics (colistin), thereby reducing the clinically used concentration of antibiotics with toxic side effects and avoiding their adverse effects on patients. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1The results are for the identification of compounds that enhance the inhibition of bacterial growth by colistin in Example 1 and the relevant test results for measuring the synergistic effect of the combined use of BMS-833923 (hereinafter referred to as BMS) and colistin on the inhibition of Escherichia coli growth by checkerboard experiment in Example 2.

[0029] Figure 2 The test results show that BMS-833923 in Example 3 promotes the bactericidal activity of colistin and that BMS in Example 4 directly kills Gram-positive bacteria without inducing drug resistance.

[0030] Figure 3 This is a graph showing the apoptosis test results of Escherichia coli treated with colistin and BMS-833923 in Example 5.

[0031] Figure 4 The image shows the apoptosis test results of Staphylococcus aureus when BMS-833923 was used alone in Example 5. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0033] The following provides a detailed description of the applications and pharmaceutical uses of BMS-833923 and its derivatives provided in this application.

[0034] This application proposes an application of BMS-833923 and its derivatives, specifically using BMS-833923 and its derivatives as colistin adjuvants. BMS-833923 is a known substance, commonly used as a Hedgehog pathway inhibitor. The BMS-833923 derivatives claimed in this application can be found in existing technologies; their structures will not be described in detail here.

[0035] This application utilizes BMS-833923 and its derivatives as adjuvants for colistin, which can sensitize pathogens to antibiotics (colistin), expand the therapeutic index of colistin, and allow lower, non-toxic doses of colistin to effectively kill bacterial pathogens. This not only alleviates the antibiotic resistance crisis but also effectively reduces side effects on patients.

[0036] For reference, BMS-833923 and its derivatives provided in this application can be used in combination with colistin for the treatment of bacteria.

[0037] The bacteria are mainly Gram-negative, preferably Gram-negative bacteria in the logarithmic growth phase.

[0038] In an optional implementation, 1×MIC 50 Colistin-treated Gram-negative bacteria are treated with 5-15 μg / mL of BMS-833923 and its derivatives, preferably 1×MIC. 50 Add 10 μg / mL of BMS-833923 and its derivatives to colistin-treated Gram-negative bacteria.

[0039] For reference, BMS-833923 and its derivatives, together with colistin, can be used for the combined inhibition or elimination of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and at least one of the pan-drug-resistant strains BAA-1800, BAA-1794, and BAA-1792.

[0040] It should be noted that BMS-833923 and its derivatives exhibit a synergistic effect on the growth inhibition of *E. coli* when used in combination with colistin. In contrast, this effect was not observed between BMS-833923 and its derivatives and the β-lactam antibiotic ampicillin, the quinolone antibiotic norfloxacin, or the aminoglycoside antibiotic kanamycin. The enhancing effect of BMS-833923 and its derivatives on the antibacterial activity of colistin has also been observed in other pathogens, including *Klebsiella pneumoniae*, *Acinetobacter baumannii*, and *Pseudomonas aeruginosa*, where BMS-833923 and its derivatives can enhance the effect of colistin by up to 8-512 times through synergistic action. This indicates that BMS-833923 and its derivatives can enhance the antibacterial activity of colistin.

[0041] In addition, this application also provides the application of BMS-833923 and its derivatives for the independent inhibition or elimination of Gram-positive bacteria.

[0042] Gram-positive bacteria may include at least one of Staphylococcus aureus and Bacillus subtilis, and may also include other Gram-positive bacteria.

[0043] It should be noted that, unlike its effect on Gram-negative bacteria, BMS-833923 and its derivatives can directly inhibit the growth of Gram-positive bacteria without the assistance of other antibiotics. In other words, BMS-833923 and its derivatives can directly kill Gram-positive bacteria without inducing drug resistance.

[0044] Continuing from the above, the compound BMS-833923 provided in this application can enhance the activity of colistin against various Gram-negative bacteria by up to 8-512 times. Treatment of logarithmically growing bacteria with low concentrations of colistin and BMS-833923 and its derivatives can rapidly and effectively eliminate bacteria. Notably, BMS-833923 and its derivatives can directly kill Gram-positive bacteria without the need for co-administration with any other antibiotics. Moreover, no significant drug-resistant mutants were generated in bacteria after 30 days of continuous passage in the presence of BMS-833923 and its derivatives. Preliminary mechanistic studies revealed that bacteria killed by BMS-833923 and its derivatives exhibited characteristics of bacterial apoptosis, including significantly increased phosphatidylserine (PS) exposure, DNA breaks, chromosome condensation, membrane depolarization, and caspase-like protein binding activity. In other words, BMS is an effective adjuvant that can expand the therapeutic index of colistin, allowing for the clinical use of lower, non-toxic doses of colistin for the effective treatment of drug-resistant bacterial infections.

[0045] Furthermore, this application also provides an antibacterial drug whose active ingredients include BMS-833923 and its derivatives and colistin.

[0046] The aforementioned antibacterial drugs may be, for example, drugs that inhibit or eliminate at least one of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, and pan-drug-resistant strains BAA-1800, BAA-1794, and BAA-1792.

[0047] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0048] Strains and reagents used in the examples: The strains used in the following examples are listed in Table 1. All antibiotics were purchased from Sigma-Aldrich. The apoptosis compound library (catalog number L3300) was purchased from SelleckChem. MHB and LB media were purchased from Becton Dickinson (BD). Compound BMS-833923 was purchased from Shanghai DC Chemicals. The FITC Annexin V Apoptosis Detection Kit (catalog number 556547) and APO-DIRECT™ Kit (catalog number 556381) were purchased from BD. CaspACE TM FITC-VAD-FMK In SituMarker (catalog number G7401) was purchased from Promega. DiBAC4 membrane potential assay reagent (catalog number B438) was purchased from Invitrogen. Hoechst 33342 (catalog number H1339) was purchased from Thermo Fisher Scientific.

[0049] Table 1 Bacterial strains

[0050] strains describe Gram-positive Staphylococcus aureus ATCC 25904 wild type Staphylococcus aureus ATCC 43300 MRSA Bacillus subtilis 168 (trpC2) wild type Gram-negative Escherichia coli MG1655 wild type Escherichia coli BW25113 wild type Escherichia coli MG1655 / pACYC184-mcr-1 mcr-1 positive Escherichia coli Y8 Urinary tract pathogens Vibrio cholerae SCE223 wild type Vibrio alginolyticus ATCC33787 wild type Citrobacter DBS100 wild type Salmonella 13076 wild type Vibrio parahaemolyticus RIMD 2210633 clinical Pseudomonas aeruginosa PAO1 wild type Klebsiella pneumoniae ATCC13883 Type strain Acinetobacter baumannii ATCC 19606 Type strain Acinetobacter baumannii ATCC 17978 wild type Acinetobacter baumannii ATCC BAA-1791 Multidrug resistance Acinetobacter baumannii ATCC BAA-1800 Multidrug resistance Acinetobacter baumannii ATCC BAA-1794 Multidrug resistance Acinetobacter baumannii ATCC BAA-1792 Multidrug resistance

[0051] Example 1

[0052] Identification of compounds that enhance the inhibitory effect of colistin on bacterial growth

[0053] The effect of the compound in combination with colistin on bacteria was tested by measuring bacterial growth in 96-well plates, and the compound was found to enhance the inhibition of bacterial growth by low concentrations of colistin.

[0054] A total of 689 compounds were tested.

[0055] Add 1 / 4 of the minimum inhibitory concentration (MIC) to the bacterial culture medium 50 Colistin and a single compound at 40 μM were administered, and growth was examined after 16 hours of bacterial culture. OD was set. 600 =0.2 is the critical value; identify the OD that leads to bacterial growth. 600 Compounds with a concentration <0.2 should be further investigated (e.g.) Figure 1 As shown in A), a total of 17 compounds were identified in the preliminary screening (as shown in Table 1).

[0056] When the final concentration of the compounds was further reduced to 20 μM, 15 of the 17 compounds still completely inhibited bacterial growth (OD) in the presence of 1 / 4 MIC of colistin. 600 <0.2).

[0057] Furthermore, further results showed that five of these compounds could be reacted with 1 / 8 MIC 50 Colistin combinations are completely effective in inhibiting bacterial growth. Among them, BMS-833923 (XL139, BMS) is a human SMO receptor inhibitor of the Hedgehog signaling pathway (chemical formula as shown). Figure 1 As shown in Figure B, BMS possesses potential activity in treating advanced or metastatic cancers. Clinical trials have demonstrated that the drug is well-tolerated by subjects, proving its good safety profile. Therefore, BMS has the potential for clinical use.

[0058] The relevant culture and detection methods are as follows:

[0059] ① Bacterial growth culture:

[0060] For most experiments, single colonies were picked and placed in 2 mL of MHB medium and incubated overnight at 37°C and 220 rpm. Then, the culture was diluted 1:100 to 25 mL of fresh MHB and continued. Before treatment, bacteria were cultured to mid-log phase (OD).600 =0.35~0.45 (approximately 2 hours). OD was measured using a SpectraMax M5 microplate reader (Molecular Devices). 600 Measurement. The colony forming units (CFU) / mL measurement method is as follows: 200 μL of bacterial culture was collected at each time point, washed twice with sterile 1×PBS, pH 7.2 (Santa Cruz Biotechnology), and then serially diluted in 1×PBS. 5 μL of each dilution was dropped onto LB agar plates and incubated overnight at 37°C. Only dilutions producing 20–100 colonies were counted, and the CFU / mL value was calculated using the following formula: CFU / mL = [colony count × dilution factor] / (volume in mL). Each experiment was independently repeated three times, and the mean and standard deviation of the three experimental data were calculated.

[0061] ② High-throughput screening method for colistin adjuvant:

[0062] Reference for the high-throughput screening method for colistin adjuvant: An WF, Tolliday NJ: Introduction: cell-based assays for high-throughput screening. Methods MolBiol 2009, 486: 1-12. In short, *E. coli* MG1655 was cultured in LB medium to OD using the above method. 600 =0.4. Then, 2 μL of the compound (2 mM concentration) was aliquoted into 96-well plates containing 48 μL of LB medium. Next, the bacterial culture was diluted to OD using LB medium. 600 =0.04 and colistin was added to a concentration of 1 μg / mL. 50 μL of the diluted bacteria containing 1 μg / mL colistin was added to a 96-well plate containing the compound. The final concentrations of each component in a 100 μL mixture are as follows: OD of diluted bacteria 600 =0.02, 0.5 μg / mL colistin, 40 μM compound, 2% DMSO. Four negative controls were set up in wells A12, B12, C12, and D12 by adding 2 μL of pure DMSO and the same number of bacteria. Four positive controls were set up in wells E12, F12, G12, and H12 by adding norfloxacin to a final concentration of 1 μg / mL and the same number of bacteria. OD was read immediately after each compound was added. 600 The 96-well plate was incubated at 37°C for 24 hours, and the OD was read again. 600It is best to shake the plate for 5 seconds before taking readings to obtain more accurate data. The effectiveness of the high-throughput screening method is evaluated by calculating the Z′ value. Data are standardized using a method based on quartile means. If the OD value of a compound in a well is below 0.2, the compound is selected as the “primary hit” and further validated.

[0063] Example 2

[0064] The synergistic effect of BMS and colistin on the inhibition of Escherichia coli growth was measured using a checkerboard assay.

[0065] The results showed that the combined use of BMS and colistin exhibited a significant synergistic effect (e.g., Figure 1 Figure C shows the checkerboard analysis results of the synergistic antibacterial effect of BMS and colistin against Escherichia coli MG1655. In contrast, this effect was not observed between BMS and the β-lactam antibiotic ampicillin, the quinolone antibiotic norfloxacin, or the aminoglycoside antibiotic kanamycin (data not shown).

[0066] BMS has also been observed to enhance the inhibitory effect of colistin on other pathogens, including Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. BMS can enhance the effect of colistin by up to 8 to 512 times through synergistic action (e.g., ...). Figure 1 (As shown in D). The results showed that BMS can enhance the antibacterial activity of colistin.

[0067] The relevant testing methods are as follows:

[0068] Minimum inhibitory concentration (MIC) determination and Checkerboard assay were performed according to the Clinical Laboratory Standards Institute (CLSI) guidelines. In short, the bacterial strain was cultured in MHB medium to OD using the methods described above. 600 =0.4. Then BMS and antibiotics were diluted twofold in MHB, and the concentration in MHB was approximately 1.5 × 10⁻⁴. 6 A bacterial suspension of CFU / mL was mixed in a 96-well microtiter plate. After incubation at 37°C for 24 h, the MIC was defined as the lowest concentration of BMS or the antibiotic that inhibited bacterial growth. When the exact MIC value could not be determined, such as when BMS acted alone on Gram-negative bacteria, twice the highest concentration tested was considered the MIC value. The fractional inhibitory concentration index (FICI) was calculated using the following formula: FICI = A / MIC A +B / MIC B Where A and B are the MIC values ​​of the two drugs combined to inhibit bacterial growth, respectively. A and MIC BThis is the MIC value when the drug inhibits bacteria alone. Synergistic effect is defined as FICI ≤ 0.5.

[0069] The MIC and other related tests involved in the following embodiments refer to Embodiment 2, and will not be repeated hereafter.

[0070] Example 3

[0071] BMS enhances the bactericidal activity of colistin.

[0072] To demonstrate that the synergistic inhibitory effect of BMS on colistin can be converted into an enhanced bactericidal effect on colistin, the bactericidal kinetics of colistin in the presence of BMS were investigated. Figure 2 As shown in A, 1×MIC was used to treat E. coli in the exponential phase. 50 The bactericidal effect of colistin, 10 μg / ml BMS, or a combination of both on Escherichia coli was studied by treating the bacteria.

[0073] Treatment of logarithmic-phase bacteria with colistin and BMS alone had little effect on their survival. However, BMS effectively enhanced the bactericidal activity of colistin. When used in combination, almost no surviving bacteria could be detected in the treated culture one hour after treatment. Furthermore, BMS could restore drug sensitivity to colistin-resistant bacteria. Treatment of MG1655 containing mcr-1 with colistin and BMS together effectively eliminated logarithmically growing resistant bacteria within 2 hours, while colistin alone had little effect on bacterial survival (e.g., ...). Figure 2 (as shown in B).

[0074] Similarly, the enhancing effect of BMS on colistin bactericidal activity was also detected in Acinetobacter baumannii, and the results showed that at 1×MIC... 50 Adding 10 μg / ml BMS to colistin-treated bacteria resulted in the rapid elimination of various bacterial strains, including pan-drug-resistant strains BAA-1800, BAA-1794, and BAA-1792 (e.g., Figure 2 As shown in C, where the value of CT+BMS is 0).

[0075] In summary, these results indicate that BMS can enhance the bactericidal activity of colistin, and that in the presence of BMS, lower concentrations of colistin are sufficient to eliminate bacteria, thereby achieving a bactericidal effect consistent with that of higher concentrations of colistin.

[0076] Example 4

[0077] BMS directly kills Gram-positive bacteria without inducing drug resistance.

[0078] Unlike its effect on Gram-negative bacteria, BMS can directly inhibit the growth of Gram-positive bacteria without the need for other antibiotics. The MIC of BMS alone against Gram-negative bacteria (such as Escherichia coli or Acinetobacter baumannii) is... 50 Above 100 μg / mL. However, the MIC of BMS against Staphylococcus aureus and Bacillus subtilis is... 50 The concentrations were 5 and 2.5 μg / mL, respectively (as shown in Table 2).

[0079] Table 2 shows the MICs of different strains as determined by BMS. 50

[0080] strains BMS MIC (μg / mL) Gram-positive Staphylococcus aureus ATCC 25904 (Non-MRSA) 5 Staphylococcus aureus ATCC 43300 (MRSA) 5 Bacillus subtilis 168 (trpC2) 2.5 Gram-negative Escherichia coli MG1655 ＞100 Escherichia coli BW25113 ＞100 Acinetobacter baumannii ATCC 17978 ＞100 Acinetobacter baumannii ATCC BAA-1800 (multidrug-resistant) ＞100

[0081] In addition, treating logarithmic-phase Staphylococcus aureus ATCC 25904 or Staphylococcus aureus ATCC 43300 (MRSA) with 10 μg / mL BMS for 8 hours resulted in the removal of the bacteria from the culture (e.g., Figure 2 (as shown in D and 2E).

[0082] To further investigate the bactericidal effect of BMS, an attempt was made to isolate a mutant resistant to BMS. Following the previous protocol, Staphylococcus aureus ATCC 25904 was subjected to different concentrations of BMS (0.25–4 times the MIC). 50 The bacteria were passaged for 30 days to determine the potential for Staphylococcus aureus to evolve phenotypic resistance to BMS.

[0083] Data shows that no BMS-resistant mutants (such as Staphylococcus aureus) appeared after 30 days of continuous culture. Figure 2 (As shown in F). In contrast, bacterial resistance to norfloxacin develops rapidly, and the MIC of the cultured bacteria... 50 It increased more than 400 times during this period (e.g.) Figure 2 (as shown in F).

[0084] The data above indicates that bacteria cannot evolve resistance to BMS without losing their physiological adaptations.

[0085] The relevant testing methods are as follows:

[0086] Drug resistance formation assay:

[0087] To develop resistance through continuous subculturing, Staphylococcus aureus ATCC25904 in the exponential growth phase was diluted 1:100 in 1 mL of MHB medium containing different concentrations of BMS, with different concentrations of norfloxacin used as positive controls. The bacteria were incubated at 37°C and 200 rpm and subcultured every 24 h in different concentrations (0.25, 0.5, 1, 2, 4 × MIC) of BMS or norfloxacin. The second highest allowable concentration (OD) was used for growth. 600 Cultures with ≥1% concentration were washed once with 1 ml of PBS and then diluted 1:100 with the corresponding antimicrobial agents containing different concentrations (0.25, 0.5, 1, 2, 4 × MIC) for further subculturing. If changes in MIC values ​​were observed, the concentration of the corresponding antimicrobial agent was adjusted for subsequent subculturing. This continuous subculturing was repeated for 30 days and performed three independent replicates. MIC changes were calculated by dividing the daily MIC by the initial MIC value on day 1.

[0088] Example 5

[0089] BMS-induced bacterial death exhibits characteristics similar to apoptosis.

[0090] Microscopic studies were conducted to compare changes in bacteria treated with colistin in the presence and absence of BMS. Low concentrations of colistin (1×MIC) were used. 50 Escherichia coli was treated alone or in combination with BMS, and then the treated bacteria were stained with Hochest 33342 to study the morphological changes of bacterial chromosomes.

[0091] The results showed that when bacteria were treated with a combination of colistin and BMS, most bacterial cells exhibited strong fluorescence in the center (e.g., Figure 3 As shown in Figure A, this indicates that the bacterial chromosomes have aggregated. This observed morphological change is similar to previously reported bacterial apoptosis. Therefore, it is speculated that BMS may enhance bacterial killing by promoting bacterial apoptosis.

[0092] Flow cytometry was used to detect whether the treated bacteria exhibited other typical characteristics of bacterial apoptosis.

[0093] Escherichia coli was treated with colistin or a combination of colistin and BMS, and four bacterial apoptosis markers were detected: phosphatidylserine (PS) exposure was detected using Annex V; DNA breaks were detected using TUNNEL; bacterial cell membrane depolarization was detected using BiDAC4; and caspase-like substrate binding capacity was detected using FITC-Z-VD-FMK.

[0094] The results showed that when E. coli was treated with colistin or BMS alone, very few bacteria were detected that were positive for these four markers. However, when E. coli was treated with colistin and BMS in combination, the proportion of bacterial cells positive for the four markers in the population increased significantly, reaching 30% to 90% (e.g., ...). Figure 3 As shown in Figures B to 3E, the vertical axis represents 10 μg / ml BMS and 1×MIC, respectively. 50 Annexin V, FITC-VAD-F-FMK, and DiBAC4 in Escherichia coli MG1655 were treated with colistin (1 μg / ml) for 4.5 h. (3) (and percentage of TUNNEL-positive bacterial cells).

[0095] Consistent with BMS's ability to kill Gram-positive bacteria alone, significant bacterial chromosome condensation and aggregation were also observed in Staphylococcus aureus treated with BMS alone (e.g., Figure 4 (As shown in A). Flow cytometry analysis showed that the number of Annex V, TUNNEL, BiDAC4, and FITC-Z-VD-FMK positive bacteria in BMS-treated Staphylococcus aureus was significantly increased compared with the control group (e.g., ...). Figure 4 B to Figure 4 As shown in Figure E, the vertical axis represents the levels of Annexin V, FITC-VAD-F-FMK, and DiBAC4 in Staphylococcus aureus ATCC25904 treated with 10 μg / ml BMS for 4.5 h. (3) (and percentage of TUNNEL-positive bacterial cells).

[0096] The results indicate that BMS enhances bacterial killing power by promoting bacterial apoptosis.

[0097] The relevant testing methods are as follows:

[0098] ① Flow cytometry detection of phosphatidylserine exposure:

[0099] For the detection of phosphatidylserine (PS) exposure, bacteria were cultured as described above and treated with a specified concentration of the drug. Annexin V staining was performed using the FITC Annexin V Apoptosis Detection Kit (BD Pharmingen™, catalog number 556547). PI was used as a counterstain to identify dead cells.

[0100] At each time point following drug treatment, approximately 10 samples were collected by centrifugation at 8K for 5 min at 4°C. 5Collect cells and wash twice with 1 mL of cold 1×PBS. Then resuspend the sample in 100 μL of 1× binding buffer (kit component). Next, treat each sample with 5 μL of FITC-conjugated Annexin V and 5 μL of LPI (both kit components) and incubate in the dark at room temperature (25°C) for 15 minutes. After incubation, add 400 μL of 1× binding buffer to each sample and analyze the samples by flow cytometry within 1 hour.

[0101] Fluorescence analysis was performed using a CytoFLEX flow cytometer (Beckman Coulter Life Sciences) equipped with a 488 nm argon laser for excitation and fluorescence filters at 525 ± 40 nm (FITC channel for FITC-mediated fluorescence) and 585 ± 42 nm (PE channel for PI-mediated fluorescence) for detection. The following PMT voltages were used for detection: 77 (FSC), 72 (SSC), 162 (FITC), and 192 (PI). To eliminate interference from cell debris, a primary threshold of FSC-H > 100 and a secondary threshold of SSC-H > 1000 were set. At least 10,000 cells were collected for each sample. Annexin V-positive cells were analyzed using the FITC channel, and Annexin V-positive cells were analyzed using the PE channel to determine whether they were still viable (PI-negative) or dead (PI-positive). Experiments were independently repeated three times, and data were processed and images generated using GraphPadPrism8.

[0102] ② TUNEL assay and flow cytometry for DNA fragmentation detection:

[0103] For the detection of DNA fragmentation, bacteria were cultured as described above and treated with a specified concentration of the drug. TUNEL assay was performed using the APO-DIRECT™ kit (BD Biosciences, catalog number 556381). This kit uses FITC-conjugated deoxyuridine triphosphate (FITC-dUTP) to stain DNA fragments and PI as a counterstain.

[0104] At each time point following drug treatment, approximately 10 samples were collected by centrifugation at 8K for 5 min at 4°C. 5 Collect cells and wash twice with 1 mL of cold 1×PBS. Then resuspend the sample in 1 mL of 4% paraformaldehyde (Santa Cruz Biotechnology) and incubate the cell suspension on ice for 30–60 min. Centrifuge the cells at 8 K for 5 min at 4 °C, wash once, and resuspend in 300 μL of cold 1×PBS. Add 1 mL of ice-cold 70% (v / v, in dH2O) ethanol to each sample and store the samples overnight at -20 °C.

[0105] For staining, samples were centrifuged at 8K for 5 min at 4°C, and ethanol was removed by aspiration. Cells were washed twice with 1 mL of wash buffer (kit component) and then resuspended in 50 μL of DNA-labeled solution (prepared as described in the kit). Cells were incubated in a temperature-controlled shaking incubator at 37°C and 300 rpm in the dark for 60 min. At the end of incubation, 1 mL of wash buffer (kit component) was added to each sample, and the samples were centrifuged at 8K for 5 min. Cells were washed again with 1 mL of wash buffer and resuspended in 500 μL of PI / RNase A solution (adjust the PI / RNase Staining Buffer volume to 0.3 mL if cell density is low). Samples were incubated at room temperature in the dark for 30 min. Finally, 500 μL of 1×PBS was added to dilute the samples and they were analyzed by flow cytometry.

[0106] Fluorescence analysis was performed using a CytoFLEX flow cytometer (Beckman Coulter Life Sciences). Parameter settings were the same as those used for PS exposure detection. The experiment was independently repeated three times, and data were processed and images generated using GraphPad Prism8.

[0107] ③ Observation of chromosome aggregation using fluorescence microscopy:

[0108] For observation of chromosome aggregation, bacteria were cultured as described above and treated with a specified concentration of the drug. Chromosomes were fluorescently stained using the DNA-specific dye Hoechst 33342 (Invitrogen, catalog number H1339), dissolved in deionized water to prepare a 10 mg / mL (16.23 mM) stock solution. PI was used as a counterstain and dissolved in deionized water to prepare a 1 mg / mL (1.5 mM) stock solution.

[0109] At each time point following drug treatment, approximately 10 samples were collected by centrifugation at 8K for 5 min at 4°C. 5Cells were collected and washed twice with 1 mL of cold 1×PBS. Cells were resuspended in 1 mL of filtered 1×PBS, and 1 μL of 10 mg / mL Hoechst 33342 and 1 mg / mL PI were added to each sample. The samples were incubated at room temperature in the dark for 30 minutes. After incubation, cells were spotted onto a glass slide and analyzed using a Carl Zeiss Axio Observer with an APOTOME fluorescence microscope equipped with a CoolSnap HQ CCD camera (Roper Scientific) and operated using IPLab software (Scanalytics). For fluorescence imaging, cells were analyzed using UV / 488 nm dual excitation, and fluorescence signals were measured using standard Hoechst and PI filters.

[0110] ④ Flow cytometry detection of membrane depolarization:

[0111] For the detection of membrane depolarization, bacteria were cultured as described above and treated with a specified concentration of drug. DiBAC4 was used. (3) (Invitrogen, catalog number B438) was used for cell staining, and the solution was dissolved in 70% ethanol (v / v) to prepare a 1 mg / mL stock solution. DiBAC4 (3) DiBAC4 is a membrane potential (ΔΨ) sensitive dye that can be used to monitor membrane depolarization in drug-treated cells. (3) It can penetrate depolarized but still intact cell membranes and bind to intracellular lipid components, exhibiting an enhanced fluorescent signal.

[0112] For DiBAC4 (3) Staining was performed at each time point after drug treatment, and approximately 10 samples were collected by centrifugation at 8K for 5 mins at 4°C. 5 Collect cells and wash twice with 1 mL of cold 1×PBS. Resuspend the cells in 1 mL of filtered 1×PBS and add 1 μL of 1 mg / mL DiBAC4 to each sample. (3) Incubate at room temperature in the dark for 10 minutes. After incubation, wash cells three times with 1 mL of filtered 1×PBS. Resuspend the sample in 1 mL of filtered 1×PBS and analyze using a CytoFLEX flow cytometer (Beckman Coulter Life Sciences). Parameter settings were the same as those used for PS exposure assays. The experiment was independently repeated three times, and data were processed and images generated using GraphPad Prism8.

[0113] ⑤ Flow cytometry detection of bacterial caspase-like proteins:

[0114] Caspase is considered a core component of the mechanism responsible for apoptosis in eukaryotic cells. To detect the expression of caspase-like proteins in drug-treated bacteria, CaspACE was used. TM FITC-VAD-FMK in situ labeled dye (Promega, catalog number G7462) is a fluorescent analog of the pan-caspase inhibitor Z-VAD-FMK.

[0115] For the caspase-like protein detection assay, bacteria were cultured as described above. When the bacteria grew to the mid-log phase (OD) in MHB... 600 When the concentration is 0.4%, treat with a specified concentration of the drug, and simultaneously add CaspACE at a ratio of 1:1000. TM FITC-VAD-FMK In Situ Marker (final concentration 5 μM). Samples were protected from light and incubated at 37°C and 220 rpm. At each time point after drug treatment, approximately 10 samples were collected by centrifugation at 8K for 5 min at 4°C. 5 Cells were collected and washed twice with 1 mL of cold 1×PBS. The sample was resuspended in 1 mL of filtered 1×PBS and analyzed using a CytoFLEX flow cytometer (Beckman Coulter Life Sciences). Parameter settings were the same as those used for PS exposure assays. The experiment was independently repeated three times, and data were processed and images generated using GraphPad Prism8.

[0116] In summary, the BMS identified in this study can effectively enhance the activity of colistin and reduce its effective concentration for killing bacterial pathogens. This enhanced bactericidal activity is likely achieved by activating bacterial apoptosis. Considering the proven safety of BMS in clinical trials, BMS has the potential to serve as an adjuvant for colistin.

[0117] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibacterial drug, characterized in that, The active ingredients of the antibacterial drug include BMS-833923 and colistin.

2. The antibacterial drug according to claim 1, characterized in that, The antibacterial drug is an agent that inhibits or eliminates at least one of the following: Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, and pan-drug-resistant strains BAA-1800, BAA-1794, and BAA-1792.

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