Application of antithrombotic small molecule compound BPTU as colistin adjuvant to synergistically resist gram-negative bacteria

Abstract

The invention relates to the technical field of biological medicine, in particular to application of an antithrombotic small molecule compound BPTU (CAS number 870544-59-5) as a colistin adjuvant to synergistically resist gram-negative bacteria, and the antithrombotic small molecule compound BPTU comprises a compound as shown in a formula I; according to the application of the antithrombotic small molecule compound BPTU serving as the colistin adjuvant to synergistically resist gram-negative bacteria, the BPTU and the colistin have a synergistic antibacterial effect on the gram-negative bacteria, an extracellular membrane of the gram-negative bacteria can play a role in a drug barrier, the colistin has membrane breaking activity, the barrier effect of the cell membrane is broken by the colistin, and the effect of the drug barrier is improved. The antithrombotic micromolecular compound BPTU and colistin are combined to assist the BPTU to enter cells to exert antibacterial activity, so that the antithrombotic micromolecular compound BPTU and colistin are combined for medication and have a wide application prospect in treatment of gram-negative bacteria.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, and particularly relates to the use of the anti-thrombotic small molecule compound BPTU as a colistin adjuvant to synergistically combat Gram-negative bacteria. Background Art

[0002] The 1950s was the "golden age" of antibiotic discovery. Antibiotics for treating various infections, such as erythromycin, vancomycin, and metronidazole, were successively discovered. However, few new antibiotics have been discovered since then. The development of new antibiotics is difficult, costly, and time-consuming. Therefore, the development of potentiators (adjuvants) for existing antibiotics has become one of the key topics to overcome bacterial drug resistance.

[0003] With the increasing use of polymyxin, combined with plasmid-mediated colistin resistance, it seriously undermines the last line of defense against multi-drug resistant Gram-negative pathogens and indicates the emergence of true pan-drug resistant infections. Colistin resistance is usually generated through the covalent modification of lipid A with cationic residues such as phosphoethanolamine, which is mediated by MCR-1, reducing the affinity of polymyxin for lipopolysaccharide. Therefore, new strategies are needed to address the rapidly decreasing treatment options for Gram-negative bacterial infections.

[0004] The anti-thrombotic small molecule compound BPTU was initially reported to reduce platelet aggregation and thus has anti-thrombotic function. Based on its effective biological activity and safety, scientists have deeply studied its action targets in mammals. It was found that BPTU is a non-nucleotide allosteric antagonist of the P2Y1 purinergic receptor, which is the earliest discovered and cloned member of the P2Y receptor family, belonging to the G protein-coupled receptor family, and its endogenous agonists are mainly ADP and ATP. Currently, there is no report on the antibacterial activity of BPTU against Gram-negative bacteria. Based on the current drug use strategy, exploring more types of potentiators for existing antibiotics is of great significance in clinical drug applications. Summary of the Invention

[0005] The purpose of this application is to provide the use of the anti-thrombotic small molecule compound BPTU as a colistin adjuvant to synergistically combat Gram-negative bacteria, aiming to solve the problem in the prior art that colistin alone cannot cope with the infection of colistin-resistant Gram-negative bacteria.

[0006] To achieve the above application purpose, the technical solution adopted in this application is as follows: In the first aspect, this application provides the use of an anti-thrombotic small molecule compound BPTU as a colistin adjuvant to synergistically combat Gram-negative bacteria, wherein the anti-thrombotic small molecule compound BPTU includes the compound shown in Formula I:

[0007] Formula I.

[0008] In some embodiments, the antithrombotic small molecule compound BPTU needs to be used in combination with colistin to achieve antibacterial effects against Gram-negative bacteria.

[0009] In some embodiments, the Gram-negative bacteria include at least one of Escherichia coli, Salmonella, Klebsiella pneumoniae, Enterobacter asburiae, Pantoea dispersa, and Citrobacter koseri.

[0010] In some embodiments, the Gram-negative bacteria include Gram-negative bacteria containing the colistin resistance gene mcr-1 thereof.

[0011] In some embodiments, the Gram-negative bacteria containing the colistin resistance gene mcr-1 include at least one of Escherichia coli, Salmonella, and Klebsiella pneumoniae.

[0012] In a second aspect, the present application provides an antibacterial drug for Gram-negative bacteria. The antibacterial drug includes colistin and a colistin adjuvant. Among them, the colistin adjuvant is the antithrombotic small molecule compound BPTU. Among them, the antithrombotic small molecule compound BPTU includes a compound shown in Formula I:

[0013] Formula I.

[0014] In some embodiments, the colistin includes at least one of polymyxin B and polymyxin E.

[0015] In some embodiments, the drug further includes a pharmaceutically acceptable excipient.

[0016] In some embodiments, the excipient includes at least one of a pH regulator, an osmotic pressure regulator, a bacteriostatic agent, a viscosity regulator, an antioxidant, a penetration enhancer, a diluent, a filler, a binder, a wetting agent, an absorption enhancer, a surfactant, a lubricant, and a stabilizer.

[0017] In some embodiments, the dosage form of the drug includes any one of an injection, a gel preparation, an ointment, and a liquid preparation.

[0018] In some embodiments, the drug further includes a compound that destroys the bacterial cell membrane.

[0019] The use of a small molecule anti - thrombus compound BPTU provided by the first aspect of the present application as a colistin adjuvant to synergistically combat Gram - negative bacteria. BPTU and colistin have a synergistic antibacterial effect against Gram - negative bacteria. The outer membrane of Gram - negative bacteria can act as a drug barrier, while colistin has membrane - disrupting activity. By disrupting the cell membrane barrier with colistin, it assists BPTU in entering the cell to exert antibacterial activity. Therefore, the combined use of the small molecule anti - thrombus compound BPTU and colistin has broad application prospects in the treatment of Gram - negative bacteria.

[0020] An antibacterial drug for Gram - negative bacteria provided by the second aspect of the present application. The provided antibacterial drug includes the small molecule anti - thrombus compound BPTU and colistin. The combined use of the two can block the survival and transmission process of colistin - resistant bacteria, becoming a new antibacterial and treatment plan, providing new ideas and approaches for the treatment of colistin - resistant Gram - negative bacterial infections. Brief Description of the Drawings

[0021] To more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the following - described drawings are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 It is the CRISPRi screening of Klebsiella pneumoniae under the drug pressure of BPTU provided by the embodiment of the present application, screening for the genetic factors of Klebsiella pneumoniae resistant to BPTU yejL and yejM The phenotypic verification of the outer membrane permeability of the strain after knocking them down and the sensitivity to the drug BPTU, and the result graph of the observation of the bacterial cell morphology; Figure 2 It is the result graph of the increased outer membrane permeability of colistin - resistant Escherichia coli under the sub - inhibitory concentration of colistin (CS) and the result of BPTU reducing the binding of HADA to the cell wall under the sub - inhibitory concentration of colistin (CS) provided by the embodiment of the present application; Figure 3 It is the result graph of the synergistic antibacterial activity of the combined use of BPTU and colistin against Escherichia coli ATCC25922 provided by the embodiment of the present application; Figure 4 It is the result graph of the synergistic antibacterial activity of the combined use of BPTU and colistin against Escherichia coli CSZ4 containing the colistin - resistant gene mcr-1 provided by the embodiment of the present application; Figure 5It is the result graph of the synergistic antibacterial activity of the combined use of BPTU and colistin against Salmonella ATCC14028 provided by the embodiments of the present application; Figure 6 It is the result graph of the synergistic antibacterial activity of the combined use of BPTU and colistin against Salmonella containing the colistin resistance gene mcr-1 ; Figure 7 It is the result graph of the synergistic antibacterial activity of the combined use of BPTU and colistin against Klebsiella pneumoniae CMG1-2 provided by the embodiments of the present application; Figure 8 It is the result graph of the synergistic antibacterial activity of the combined use of BPTU and colistin against Enterobacter asburiae 1383 provided by the embodiments of the present application; Figure 9 It is the result graph of the synergistic antibacterial activity of the combined use of BPTU and colistin against Citrobacter koseri 1232 provided by the embodiments of the present application; Figure 10 It is the result graph of the synergistic antibacterial activity of the combined use of BPTU and colistin against Pantoea dispersa 1488 provided by the embodiments of the present application; Figure 11 It is the result graph of the bactericidal curve of the combined use of BPTU and colistin against colistin-resistant Escherichia coli, Klebsiella pneumoniae, and Salmonella provided by the embodiments of the present application; Figure 12 It is the in-vivo experiment of the combined use of BPTU and colistin against colistin-resistant Escherichia coli E.coli CSZ4( mcr-1 ). Detailed implementation manners

[0023] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application clearer, the present application will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0024] In the present application, the term "and / or" describes the association relationship of associated objects and indicates that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist simultaneously, and B exists alone. Where A and B may be singular or plural. The character " / " generally represents an "or" relationship between the associated objects before and after.

[0025] In this application, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following" or a similar expression means any combination of these items, including any combination of single item or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can both represent: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or multiple respectively.

[0026] It should be understood that in various embodiments of this application, the magnitude of the sequence numbers of the above - mentioned processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of this application.

[0027] The terms used in the embodiments of this application are only for the purpose of describing specific embodiments, and are not intended to limit this application. The singular forms "a" and "the" used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0028] The weight of the relevant components mentioned in the specification of the embodiments of this application not only can refer to the specific content of each component, but also can represent the proportional relationship of the weights between components. Therefore, as long as the content of the relevant components in the specification of the embodiments of this application is scaled up or down proportionally, it is within the scope disclosed in the specification of the embodiments of this application. Specifically, the mass in the specification of the embodiments of this application can be mass units well - known in the chemical industry such as µg, mg, g, kg, etc.

[0029] The terms "first" and "second" are only used for descriptive purposes, to distinguish objects such as substances from each other, and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. For example, without departing from the scope of the embodiments of this application, the first XX can also be referred to as the second XX, and similarly, the second XX can also be referred to as the first XX. Thus, the features defined with "first" and "second" can explicitly or implicitly include one or more of such features.

[0030] The term "CS" is an abbreviation of "Colistin" and represents colistin.

[0031] The term " E.coli " is an abbreviation of " Escherichia coli " and represents Escherichia coli.

[0032] The term " S.m " is an abbreviation of " Salmonella " and represents Salmonella.

[0033] The term " K.p " means " Klebsiella pneumoniae ", indicating Klebsiella pneumoniae.

[0034] The term "FICI" means "Fractional Inhibitory Concentration Index", indicating the fractional inhibitory concentration index.

[0035] The term "HADA" means "7-hydroxycoumarin-amino-D-alanine", which is a fluorescent D-alanine derivative widely used in the study of bacterial cell wall synthesis and dynamics. Its full name is 7-hydroxycoumarin-3-carboxylic acid–D-alanine, and its structure contains a 7-hydroxycoumarin fluorescent group that emits blue light and is bound to D-alanine (D-Ala) through chemical linkage.

[0036] The term "NPN" means "N-phenyl-1-naphthylamine", which is a classical fluorescent probe widely used to detect changes in the outer membrane permeability of Gram-negative bacteria.

[0037] The term "DAPI" means "4′,6-diamidino-2-phenylindole", which is a small molecule dye that can penetrate cell membranes (especially in fixed or permeabilized cells) and specifically bind to DNA. It binds particularly easily to A-T base pairs in DNA and is therefore mainly used to stain cell nuclei or bacterial nucleic acids.

[0038] The term "Nile Red" means "9-diethylamino-5H-benzo[α]phenoxazine-5-one", which is a hydrophobic fluorescent dye that can specifically insert into lipid environments.

[0039] Currently, the situation of Gram-negative bacterial infections faced by humans is becoming increasingly severe, especially the significant increase in the spread of drug-resistant strains. These pathogens include extended-spectrum beta-lactamase (ESBL)-producing and carbapenem-resistant organisms (CRE), which are commonly found in conditions such as urinary tract infections, pneumonia, and sepsis. Drug-resistant Gram-negative bacteria show significant resistance to conventional antibiotics (such as cephalosporins and carbapenems), making treatment more complex. Colistin (also known as polymyxin E) is an "emergency last resort" antibiotic used to treat multi-drug resistant Gram-negative bacterial infections. However, in recent years, the problem of colistin resistance has become increasingly serious, especially in some key pathogens such as Escherichia coli and Acinetobacter baumannii. Resistance mainly occurs through two mechanisms: one is chromosomal mutation leading to changes in the lipopolysaccharide (LPS) structure, and the other is through plasmids carrying mcr genes, and different variants have been discovered mcr-1 up to mcr-10 . In the face of colistin resistance, various combination treatment strategies are being explored, including the combination of polymyxins with other antibiotics, such as sulfamethoxazole / polymyxin and ceftazidime / avibactam. At the same time, new antibiotics (such as cefiderocol and sulfonamide-polymyxin combinations) are also under development, although clinical data are still limited.

[0040] Based on this, in the embodiments of the present application, BPTU is used as an adjuvant for colistin, and by using them in combination, the effectiveness of colistin against Gram-negative bacteria is improved, and thus the therapeutic effect of colistin on colistin-resistant bacteria is effectively enhanced, providing new ideas and approaches for the treatment of Gram-negative bacterial infections.

[0041] In a first aspect of the embodiments of the present application, there is provided an application of an anti-thrombotic small molecule compound BPTU as an adjuvant for colistin to synergistically resist Gram-negative bacteria, wherein the anti-thrombotic small molecule compound BPTU includes a compound shown in Formula I:

[0042] Formula I.

[0043] In the application of an anti-thrombotic small molecule compound BPTU as an adjuvant for colistin to synergistically resist Gram-negative bacteria provided by the first aspect of the embodiments of the present application, BPTU and colistin have a synergistic antibacterial effect against Gram-negative bacteria. The outer membrane of Gram-negative bacteria can act as a drug barrier, and colistin has membrane-breaking activity. Colistin breaks the barrier of the outer membrane to assist BPTU in entering the cell to exert antibacterial activity. Therefore, the combined use of the anti-thrombotic small molecule compound BPTU and colistin has broad application prospects in the treatment of Gram-negative bacteria.

[0044] In some embodiments, the antithrombotic small molecule compound BPTU needs to be used in combination with colistin to achieve antibacterial effects against Gram-negative bacteria. The embodiments of the present application prove through experimental analysis that when BPTU and colistin are used in combination, at lower drug concentrations of both, a synergistic antibacterial effect against Gram-negative bacteria can be achieved. Thus, a new treatment plan for Gram-negative bacteria is provided.

[0045] In some embodiments, the Gram-negative bacteria include at least one of Escherichia coli, Salmonella, Klebsiella pneumoniae, Enterobacter asburiae, Pantoea dispersa, and Citrobacter koseri.

[0046] In some embodiments, the Gram-negative bacteria include Gram-negative bacteria containing the colistin resistance gene mcr-1 thereof.

[0047] In some embodiments, the Gram-negative bacteria containing the colistin resistance gene mcr-1 thereof include at least one of Escherichia coli, Salmonella, and Klebsiella pneumoniae. Further, when BPTU and colistin are used in combination, at lower drug concentrations of both, that is, at sub-inhibitory concentrations of colistin, the outer membrane permeability of colistin-resistant bacteria can be increased. On this basis, BPTU can cross the barrier of the bacterial outer membrane and enter the cell to bind to peptidoglycan, preventing the bacteria from forming cell walls, and thus achieving a bactericidal effect. Therefore, a synergistic antibacterial effect against Escherichia coli, Klebsiella pneumoniae, and Salmonella containing the colistin resistance gene mcr-1 thereof can be achieved. Therefore, an effective strategy for treating infections of the above-mentioned strains containing the colistin resistance gene is provided.

[0048] In addition, when BPTU and colistin are used in combination, at lower drug concentrations of both, they can play a good therapeutic role in treating infections in mice. Therefore, it further proves that the combination of BPTU and colistin has broad application prospects in clinical practice.

[0049] In the second aspect of the embodiments of the present application, an antibacterial drug for Gram-negative bacteria is provided. The antibacterial drug includes colistin and a colistin adjuvant, wherein the colistin adjuvant is the antithrombotic small molecule compound BPTU, and the antithrombotic small molecule compound BPTU includes a compound shown in Formula I:

[0050] Formula I.

[0051] The antibacterial drug for Gram-negative bacteria provided in the second aspect of the embodiments of the present application includes the antithrombotic small molecule compound BPTU and colistin. Using the two in combination can block the survival and transmission process of colistin-resistant bacteria, becoming a new antibacterial and treatment plan, providing new ideas and approaches for the treatment of colistin-resistant Gram-negative bacterial infections.

[0052] In some embodiments, colistin includes at least one of polymyxin B and polymyxin E.

[0053] In some embodiments, the drug further includes a pharmaceutically acceptable excipient.

[0054] In some embodiments, the excipient includes at least one of a pH regulator, an osmotic pressure regulator, an antibacterial agent, a viscosity regulator, an antioxidant, a penetration enhancer, a diluent, a filler, a binder, a wetting agent, an absorption enhancer, a surfactant, a lubricant, and a stabilizer.

[0055] In some embodiments, the dosage form of the drug includes any one of an injection, a gel preparation, an ointment, and a liquid preparation.

[0056] In some embodiments, the drug further includes a compound that disrupts the bacterial cell membrane.

[0057] The following is an illustration with specific embodiments.

[0058] Example 1 Screen for compounds in a small molecule compound library that can synergistically act against Gram-negative bacteria with colistin.

[0059] Purchase a small molecule compound library with product number T4132 from targetMol ( https: / / www.targetmol.cn / search?keyword=BPTU ). The combined drug sensitivity experiment of BPTU in this compound library and colistin is as follows: First, inoculate Escherichia coli ATCC25922, Salmonella ATCC14028, Klebsiella pneumoniae CMG1-2, Enterobacter asburiae, Pantoea dispersa, and Citrobacter koseri preserved in this laboratory into LB test tube broth, and incubate at 37 °C and 180 rpm for 4 h. Use an inoculation loop to dip the enriched broth and streak single colonies on an LB agar medium, and incubate overnight in a 37 °C constant temperature incubator.

[0060] 1.1 Determination of the MIC of the strain Conduct the experiment according to the operating standards of CLSI. Inoculate Escherichia coli ATCC25922, Salmonella ATCC14028, Klebsiella pneumoniae CMG1-2, Enterobacter asburiae, Pantoea dispersa, and Citrobacter koseri into fresh MH test tube broth, and incubate at 37 °C and 180 rpm for 4 h to make the bacterial liquid concentration 0.5 McFarland turbidity, and dilute 100 times to 1×10 6Reserve the bacterial count of CFU / ml. Inoculate Escherichia coli ATCC25922, Salmonella ATCC14028, Klebsiella pneumoniae CMG1-2, Enterobacter asburiae, Pantoea dispersa, and Citrobacter koseri into fresh MH test tube broth, and incubate at 37°C with 200 rpm for 6 - 8 h to make the bacterial suspension concentration reach 0.5 McFarland turbidity, then dilute it 100 times to 1×10 6 CFU / ml bacterial count for standby. The drug preparation and dilution methods are shown in Table 1, and the judgment criteria for antibiotic susceptibility results are shown in Table 2.

[0061] Agar dilution method: Add 1 ml of the drug to be tested and its corresponding diluent (Table 1) into a sterile empty petri dish, then use the two-fold dilution method to dilute it into different drug concentrations, and record the drug concentration in the petri dish. After the autoclaved MH agar cools down, use a pipette to transfer 19 ml of MH agar to each petri dish. After the agar solidifies, use an inoculator to inoculate the diluted bacterial suspension onto the MH agar plates with different drug concentrations and the blank control medium. After the bacterial suspension dries at room temperature, incubate at 37°C for 18 h and then record the MIC values of different antibiotics.

[0062] Broth dilution method: Add 180 μL of MH broth to the first column of a 96-well plate, and add 100 μl of MH broth to the remaining wells. Then add 20 μL of the antibiotic with a concentration of 5120 mg / L to each well in the first column, mix well, and perform gradient dilution in a two-fold dilution manner from left to right. Finally, add 100 μL of the diluted bacterial suspension to each well, incubate at 37°C for 18 - 22 h, observe the results, and interpret the results. According to the MIC quality control range of CLSI, the Escherichia coli ATCC25922 is used as the judgment standard. If the results are consistent with the CLSI standard, it indicates that the MIC results of this experiment are reliable.

[0063] Table 1

[0064] Table 2

[0065] Table 2 refers to the 32nd edition (2022) of the "M100" standard issued by the Clinical and Laboratory Standards Institute (CLSI).

[0066] 1.2 Checkerboard assay: Using colistin as drug A and a small molecule compound as drug B, add 50 μL of fresh MH broth to columns 1 - 8 of a 96 - well plate. Dilute drug A to 16 times its MIC value (since drug A is serially diluted in the plate, the highest final concentration of drug A is 2 times the MIC), and dilute drug B to 8 times its MIC value (since drug B is diluted to the corresponding concentration outside, the highest final concentration of drug B is 2 times the MIC). Add 50 μL of drug A at 16 times the MIC value to wells 1 - 8 in row A. Use an 8 - channel pipette, set the volume range to 50 μL, and serially dilute from row A to row G, discarding the pipette tips. When drug B is diluted to 8 times the MIC value, further serially dilute it in a clean petri dish in 6 gradients with concentrations of 8 MIC value, 4 MIC value, 2 MIC value, 1 MIC value, 1 / 2 MIC value, 1 / 4 MIC value, and 1 / 8 MIC value respectively. Add these 7 gradients to rows A - H in column 1, rows A - H in column 2... to rows A - H in column 7, and add 50 μL of MH broth to rows A - H in column 8. Dilute ATCC 25922 that has grown to the logarithmic phase 100 - fold in a blank petri dish to obtain a concentration of approximately 10 6 CFU / ml, and add 100 μL to each well in columns 1 - 8. Then place it in an incubator at 37°C for 18 - 22 h and judge the results. Each experiment is repeated three times.

[0067] Result determination: In the laboratory, the judgment basis for the combined drug sensitivity test is to calculate the fractional inhibitory concentration index (FICI): FICI index = MIC of drug A in combination / MIC of drug A alone + MIC of drug B in combination / MIC of drug B alone.

[0068] Table 3

[0069] 1.3 Bactericidal experiment of BPTU combined with colistin on strains containing colistin - resistant genes mcr-1 1. Autoclaving: 100 ml of LB broth, MHA plates, 200 ml of normal saline, several 2 - ml Eppendorf tubes, 100 ml of pure water, filter membranes, several 1 - ml and 200 - μL pipette tips, and several 10 - ml Eppendorf tubes.

[0070] 2. Bacterial culture: Inoculate Escherichia coli, Klebsiella pneumoniae, and Salmonella strains containing colistin - resistant genes mcr-1 into LB test - tube broth at 9 p.m., let it stand overnight at 37°C, and then place it in a shaker at 37°C for 30 min the next morning. Measure the OD value and dilute the bacterial solution to 1×10 6 CFU / ml.​

[0071] 3. According to the experimental results of combined drug use, dilute the drugs to the corresponding concentrations with a bacterial liquid of 1×10 6 CFU / ml (concentrations of each drug group: single drug BPTU = 8.0 μg / ml, single drug CS = 2.0 μg / ml; combined drug BPTU+CS group = 8.0 μg / ml + 2.0 μg / ml; WT group). 4. After mixing the above evenly, culture 1 ml of the liquid in at least 5 ml flow tubes. This is the start of the 0 h timing. Then, perform serial dilution and plate counting at the time points of 4, 8, 12, and 24 h respectively.

[0072] 1.4 CRISPRi screening for genes related to the sensitivity of Klebsiella pneumoniae to BPTU.

[0073] For the 7th and 14th generations of the Klebsiella pneumoniae CRISPRi library, pre-induce. Thaw the library stored at -80°C and inoculate it into 4 ml of LB broth at a ratio of 1:100 and place it in a shaker at 37°C and 250 rpm for activation (OD 600 ≈0.6), then transfer it to 4 ml containing 1 mM IPTG at a ratio of 1:100 and place it in a shaker at 37°C and 250 rpm for 7th generation pre-induction (OD 600 ≈0.6) of the induced strain. Re-inoculate the 7th generation pre-induced library into 4 ml containing 1 mM IPTG at a ratio of 1:100 for 14th generation pre-induction (OD 600 ≈0.6) of the induced strain.

[0074] Screening with the 7th generation library: Take 50 μL of the 7th generation induced library and add it to 4 ml of LB broth containing 8.0 μg / ml BPTU. The grouping, treatment conditions, and genomic concentrations extracted after treatment are shown in the following table: Table 4

[0075] Screening with the 14th generation: Take 50 μL of the 14th generation induced library and add it to 4 ml of LB broth containing 8.0 μg / ml BPTU. The grouping, treatment conditions, and genomic concentrations extracted after treatment are shown in the following table: Table 5

[0076] Using the above-extracted genomic DNA as a template, amplify the sgRNA sample pool for Illumina sequencing with a high-fidelity enzyme. The primer sequences used for PCR and the PCR amplification program are as follows: Table 6: Primer sequences used

[0077] Table 7: PCR reaction system

[0078] Table 8: Reaction procedure of the above PCR

[0079] 1.5 NPN determination of knockdown strains yejM and yejL the outer membrane permeability after the gene.

[0080] Pick single colonies of CRISPRi targeting yejM 、 yejL, gfp (Nontargeting) genes from the LB agar plate, inoculate them into 1 ml of LB broth respectively, induce the yejM gene with 0.1 mM IPTG, induce the yejL gene with 0.05 mM IPTG, with 3 parallels in each group. Incubate at 37 °C and 220 rpm until OD 600 ≈ 0.3, then take out, centrifuge to remove the supernatant. Then wash the culture medium with 5 μM HEPES (pH 7.0 + 5 μM glucose), suspend, and repeat twice. Then dilute it 1:10 with HEPES (pH 7.0 + 5 μM glucose) to wash the culture medium, and finally add the dye NPN to a final concentration of 10 μM. Take 200 μl of the probe-labeled bacterial cells and add them to a 96-well black plate. After incubating for 30 min, measure the fluorescence on an Infinite M200 Microplate reader (Tecan), with the excitation wavelength at 350 nm and the emission wavelength at 420 nm.

[0081] 1.6 NPN determination of the outer membrane permeability of the treated strain under the sub-inhibitory concentration of colistin E.coli of CSZ4.

[0082] Take the E.coli CSZ4 stored at -80 °C and inoculate it into 3 ml of fresh LB broth at a ratio of 1:100 and place it in a shaker at 37 °C and 250 rpm for culture until the strain grows to OD 600 ≈ 0.6. Then inoculate it into LB broth containing different colistin concentrations at a ratio of 1:100 again, which are 0×MIC, 1 / 8×MIC, 1 / 4×MIC, 1 / 2×MIC, 1×MIC, with 3 parallels in each group. Incubate the above groups in a shaker at 37 °C and 250 rpm for culture until the strain grows to OD 600≈0.3, centrifuge to remove the supernatant. Then wash the culture medium with 5 μM HEPES (pH 7.0 + 5 μM glucose), suspend, and repeat twice. Then dilute it 1:10 into the HEPES (pH 7.0 + 5 μM glucose) washing culture medium, and finally add the dye NPN to a final concentration of 10 μM. Take 200 μl of the probe-labeled bacterial cells and add them to a 96-well black plate. After incubating for 30 min, measure the fluorescence on an Infinite M200 Microplatereader (Tecan), with an excitation wavelength of 350 nm and an emission wavelength of 420 nm.

[0083] 1.7 Microscopic observation of CRISPRi knockdown strains yejM and yejL the morphology of the strains after the gene.

[0084] Transfer the CRISPRi knockdown strains yejM and yejL the strains cultured overnight to fresh medium, add IPTG and NileRed for induction + staining and culture until the OD of the bacterial amount 600 value is about 0.6; centrifuge the above culture solution, remove the supernatant, and at the same time add 1 ml of 4% PFA fixative, keep it in the dark for 1 h; after fixation, centrifuge to remove the supernatant (2400 x g, 4 min), and resuspend it with an equal volume of the prepared QS (100 mM Tris) (remove the residual PFA); use a prefabricated mold to prepare 2% Agarose for sample loading and observation, and add the corresponding dye during preparation; drop the bacterial solution resuspended with QS onto the gel platform, cover with a cover slip, and collect images on the machine; working concentration of the dye: DAPI 1 μg / ml, Nile Rea 0.2 μg / ml, SYTOX 1:2000.

[0085] 1.8 Determination of the sensitivity of CRISPRi knockdown strains yejM and yejL the strains after the gene to BPTU and vancomycin.

[0086] Take out the CRISPRi knockdown strains yejM and yejL from the bacterial library stored at -80 °C, inoculate them 1:100 into fresh LB broth, and shake at 37 °C until the OD 600 is about 0.6; inoculate the two strains 1:100 into fresh LB broth respectively, and set to add IPTG or not add IPTG and continue to shake until the OD 600 is about 0.6, and on this basis, dilute them 1:10 successively to 10 -1 、10 -2 、10 -3 、10 -4, 10 -5 , 10 -6 gradients; 10 μL of the above-mentioned bacterial solution was respectively taken and dropped on IPTG - , IPTG + , IPTG - +BPTU + / Van + , IPTG + +BPTU + / Van + LB agar plates, the concentration of BPTU was 5 μg / ml, and vancomycin Van was 5 μg / ml. The plates with the drops were placed at 37°C for overnight culture.

[0087] Result analysis 1. The antibacterial effect results of the small molecule compound BPTU on the selected Gram-negative bacteria are shown in Table 9.

[0088] Table 9

[0089] It can be seen that the small molecule compound BPTU shown in Formula I provided has no antibacterial effect on Klebsiella pneumoniae CMG1-2, Salmonella typhimurium GP9, Salmonella typhimurium ATCC14028, Escherichia coli CSZ4, Escherichia coli ATCC25922, Enterobacter asburiae, Pantoea dispersa, Citrobacter koseri at a concentration up to 256 μg / ml.

[0090] 2. In order to explore the genetic basis of natural resistance of Gram-negative bacteria to BPTU at the whole genome level. In the examples of this application, CRISPRi screening was carried out on a clinical multi-drug resistant Kp (Klebsiella pneumoniae) strain under the pressure of BPTU. This screening uses a CRISPRi library covering the whole genome scale, which can induce knockdown of almost all genes in Kp, so as to quantify the chemical-genetic interactions of essential and non-essential genes and provide a global overview of gene-drug interactions in Kp. Under the condition of pre-inducing the library, in the examples of this application, BPTU at a concentration of 16 μg / ml was used to act on the Kp library. After about 3 hours (the OD of the bacterial solution 600 =0.6), genomic DNA was extracted from the culture, amplified with specific primers and the sgRNA (single-guide RNA) abundance was analyzed by deep sequencing. The screened genes were analyzed and identified by the 2fastq2 software. Generally speaking, in the examples of this application, 2 genes were identified whose knockdown led to the change of Kp from natural resistance to sensitivity to BPTU, and no gene knockdown led to more resistance to BPTU ( Figure 1 A). The two sensitizing genes are PCBIOHEG_01623 ( YejM), and PCBIOHEG_01624 ( yejL hypothetical protein with unknown function) ( Figure 1 of B). Among them, the inner membrane protein PCBIOHEG_01623 ( YejM ) is a metalloenzyme that regulates LPS biosynthesis ( Figure 1 of C). Under the conditions of IPTG concentrations of 100 μM and 50 μM, yejM and yejL were knocked down respectively. It can be seen that knocking down the two genes under these conditions did not affect the normal growth of the strain (such as Figure 1 of E, F). After knocking down these two genes, in the medium with BPTU and vancomycin concentrations of 5 μg / ml, the sensitivity of the strain to BPTU and vancomycin was significantly increased (such as Figure 1 of G, H). Considering that inhibiting them sensitizes Kp to BPTU, in order to clarify the mechanism by which PCBIOHEG_01623 - PCBIOHEG_01624 proteins mediate BPTU resistance in this application example, the outer membrane permeability of the strain after knocking down the above two sensitizing genes was measured using 1-N-phenylnaphthylamine (1-Naphthylphenylamine NPN). The results showed that after knocking down the two genes, the outer membrane permeability was significantly increased compared with the control group (sgRNA-non-targeted) after knocking down the two sensitizing genes (such as Figure 1 of I). In addition, the morphology after knocking down these two genes was further observed under a microscope. It can be seen that there were no morphological changes in the control group (sgRNA-non-targeted), while the sgRNA targeting PCBIOHEG_01623 and PCBIOHEG_01624 bacteria changed the bacteria from normal morphology to "spherical" (such as Figure 1 of J and K). In addition, based on the above results, it can be known that inhibiting the outer membrane of Kp can change the strain from being resistant to BPTU to being sensitive. In addition, in this application example, the outer membrane permeability of the strain after treatment with sub-inhibitory concentration of colistin was tested. The results showed that the outer membrane permeability of the strain decreased significantly after treatment with sub-inhibitory concentration of colistin (1 / 2, 1 / 4 MIC) ( Figure 2 of A). At the same time, in this application example, it was tested that under the treatment with sub-inhibitory colistin (1 / 2 MIC), BPTU could significantly reduce the ability of HADA to bind to the cell wall ( Figure 2 of B), indicating that after colistin changed the cell membrane permeability, BPTU effectively entered the cell and blocked the synthesis of the cell wall. Therefore, in this application example, BPTU and the membrane-targeting agent colistin were used in combination, and the in vitro and in vivo effects of their combination were explored.

[0091] 3. The results of the synergistic antibacterial activity of the small molecule compound BPTU and colistin in combination against Escherichia coli ATCC25922 are asFigure 3 As shown, it can be seen that when colistin is used alone, its MIC is 2.0 μg / ml. When combined with BPTU, at a BPTU concentration of 1.0 μg / ml, the MIC of the strain against colistin drops to 0.25 μg / ml. According to the above formula for combined drug use, the FICI of the two in combination is 0.1289, which is less than 0.5. Therefore, the two have a synergistic effect.

[0092] The synergistic antibacterial activity of the small molecule compound BPTU in combination with colistin against Escherichia coli mcr-1 containing the colistin resistance gene E.coli CSZ4 ( mcr-1 ) is as shown in Figure 4 As shown, it can be seen that when colistin is used alone, its MIC is 4.0 μg / ml, which is a colistin-resistant bacterium. When combined with BPTU, at a BPTU concentration of 2 μg / ml, the MIC of the strain against colistin drops to 0.5 μg / ml, becoming sensitive to colistin. According to the above formula for combined drug use, the FICI of the two in combination is 0.1328, which is less than 0.5. Therefore, the two have a synergistic effect.

[0093] The synergistic antibacterial activity of the small molecule compound BPTU in combination with colistin against Salmonella ATCC14028 is as shown in Figure 5 As shown, it can be seen that when colistin is used alone, its MIC is 1.0 μg / ml. When combined with BPTU, at a BPTU concentration of 1.0 μg / ml, the MIC of the strain against colistin drops to 0.125 μg / ml. According to the above formula for combined drug use, the FICI of the two in combination is 0.1289, which is less than 0.5. Therefore, the two have a synergistic effect.

[0094] The synergistic antibacterial activity of the small molecule compound BPTU in combination with colistin against Salmonella containing the colistin resistance gene mcr-1 is as shown in Figure 6 As shown, it can be seen that when colistin is used alone, its MIC is 4.0 μg / ml, which is a colistin-resistant bacterium. When combined with BPTU, at a BPTU concentration of 2.0 μg / ml, the MIC of the strain against colistin drops to 0.5 μg / ml, becoming sensitive to colistin. According to the above formula for combined drug use, the FICI of the two in combination is 0.2578, which is less than 0.5. Therefore, the two have a synergistic effect.

[0095] The synergistic antibacterial activity of the small molecule compound BPTU in combination with colistin against Klebsiella pneumoniae CMG1-2 is as shown in Figure 7As shown, it can be seen that when colistin is used alone, its MIC is 8.0 μg / ml, and it is a colistin-resistant bacterium; when combined with BPTU, when the concentration of BPTU is 2.0 μg / ml, the MIC of the strain against colistin drops to 1.0 μg / ml, and it becomes sensitive to colistin. According to the above formula for combined drug use, the FICI of the two in combination is 0.2578, and this value is less than 0.5. Therefore, the two have a synergistic effect.

[0096] The synergistic antibacterial activity of the small molecule compound BPTU combined with colistin against Enterobacter asburiae 1383 is as Figure 8 shown. It can be seen that when colistin is used alone, its MIC is 8.0 μg / ml, and when combined with BPTU, when the concentration of BPTU is 4.0 μg / ml, the MIC of the strain against colistin drops to 2.0 μg / ml. According to the above formula for combined drug use, the FICI of the two in combination is 0.1406, and this value is less than 0.5. Therefore, the two have a synergistic effect.

[0097] The synergistic antibacterial activity of the small molecule compound BPTU combined with colistin against Citrobacter koseri 1232 is as Figure 9 shown. It can be seen that when colistin is used alone, its MIC is 0.5 μg / ml, and when combined with BPTU, when the concentration of BPTU is 0.25 μg / ml, the MIC of the strain against colistin drops to 0.125 μg / ml. According to the above formula for combined drug use, the FICI of the two in combination is 0.2509, and this value is less than 0.5. Therefore, the two have a synergistic effect.

[0098] The synergistic antibacterial activity of the small molecule compound BPTU combined with colistin against Pantoea dispersa 1488 is as Figure 10 shown. It can be seen that when colistin is used alone, its MIC is 1.0 μg / ml, and when combined with BPTU, when the concentration of BPTU is 0.25 μg / ml, the MIC of the strain against colistin drops to 0.25 μg / ml. According to the above formula for combined drug use, the FICI of the two in combination is 0.2509, and this value is less than 0.5. Therefore, the two have a synergistic effect.

[0099] The bactericidal curves of the small molecule compound BPTU combined with colistin against colistin-resistant Escherichia coli, Klebsiella pneumoniae, and Salmonella are as Figure 11 shown. It can be seen that when colistin is 2.0 μg / ml or BPTU is 8.0 μg / ml alone, there is no significant bactericidal effect, while when colistin 2.0 μg / ml is combined with BPTU 8.0 μg / ml, a significant bactericidal effect can be seen.

[0100] Example 2 In vivo experiment of combined drug use 2.1 Evaluation of the effect of combined use of BPTU and colistin on mcr-1 positive bacterial skin infection model ① Mice: 6 - 8 weeks old, male, BaLB / C mice, weighing 20 - 25 g.

[0101] ② - 3 days: Shave hair, use depilatory cream.

[0102] ③ - 2 days: Wound test (diameter 8 mm; depth 6 mm), anesthetic; ④ - 1 day: Bacterial infection, drop on the wound (30 μL 1x10 8 CFU / ml), take wound exudate one day later, smear on agar plate to see if there are colonies to verify whether the infection model is successful; ⑤ 0 day: Start treatment.

[0103] According to the above in vitro combined drug use results, set the following experimental groups: ① Blank group (wound exposed, no treatment); ② Solvent DMSO group (100 μL, pH = 7.0); ③ BPTU group (100 μL 2.0 μg / ml); ④ Colistin group (100 μL 2.0 μg / ml); ⑤ BPTU + colistin group (100 μL 2.0 + 2.0 μg / ml); Drop the drug on the wound once a day, 50 μL each time, for 7 consecutive days.

[0104] Indicators: 1: Wound photography: Take pictures regularly every day for 7 consecutive days; count the wound healing rate; 2: Body weight change: 1 - 7 days; 3: Bacterial count: Take wound tissue on the 7th day, put it into a centrifuge tube containing 1 ml PBS, grind thoroughly, culture at 37℃ for 1 h, and smear on the plate.

[0105] Result analysis: As Figure 12 shown, Figure 12 A in it is the schematic diagram of the curative effect of BPTU combined with colistin on the mouse model infected with Escherichia coli E.coli CSZ4 ( mcr-1 positive). Figure 12 B in it is the representative photo of the skin healing process after treatment with drugs after infection; it can be seen that from the 3rd day to the 7th day after treatment, the wound of the combined drug group is smaller than that of the control group, solvent group, colistin single - drug group, and BPTU single - drug group.Figure 12 C shows the laser speckle images of the wound surfaces of each group on days 1 - 7 of treatment (within the black dashed box); it can be seen that in the combined drug treatment group... Figure 12 D shows the body weight changes of the mice on days 1 - 7 after treatment; it can be seen that the body weight of the mice in the combined drug treatment group began to increase from day 3, and the growth rate was higher than that of the other above - mentioned groups. Figure 12 E shows the calculation of the wound area using ImageJ software, and the data of the lesion sizes of each treatment group on day 7 were statistically analyzed. The black dashed line represents the 50% and 80% healing rates; it can be seen that on day 3 after treatment, the wound healing rate of the combined drug group reached 50%, and on day 6 of treatment, it reached 80%, while the other above - mentioned groups did not reach the same healing rate as the combined drug group within the same treatment time. Figure 12 F shows that BPTU reduces the bacterial load of skin wound infections; it can be seen that the mean bacterial load of the mice in the combined treatment group was below 1×10 4 CFU / ml, while the bacterial loads of the other above - mentioned groups were all close to 1×10 7 CFU / ml. In the skin model of this application example, the number of CFUs of Escherichia coli colonized in the wound on day 7 after infection was calculated. E.coli CFU of CSZ4. P The values were analyzed by one - way ANOVA. Each bar represents the mean ± SD of 5 independent biological replicates, P The values were <0.05 and <0.01 respectively, and were represented by "*" and "**" respectively.

[0106] In summary, the application of an antithrombotic small - molecule compound BPTU as a colistin adjuvant to synergistically resist Gram - negative bacteria provided in the embodiment of this application. BPTU and colistin have a synergistic antibacterial effect on Gram - negative bacteria. The outer membrane of Gram - negative bacteria can play a role in drug barrier, while colistin has membrane - disrupting activity. Colistin breaks the barrier of the outer membrane to assist BPTU in entering the cell to exert antibacterial activity. Therefore, the combined use of the antithrombotic small - molecule compound BPTU and colistin has broad application prospects in the treatment of Gram - negative bacteria.

[0107] The above are only the preferred embodiments of this application, and are not intended to limit this application. Any modifications, equivalent replacements, and improvements made within the spirit and principle of this application shall be included within the protection scope of this application.

Claims

1. An application of an antithrombotic small molecule compound BPTU as a colistin adjuvant to synergistically fight Gram-negative bacteria, wherein: The antithrombotic small molecule compound BPTU includes a compound as shown in Formula I: Formula I.

2. The use according to claim 1, characterized in that: The antithrombotic small molecule compound BPTU needs to be used together with colistin to achieve the antibacterial effect on Gram-negative bacteria.

3. The use according to claim 1 or 2, characterized in that: The Gram-negative bacteria include at least one of Escherichia coli, Salmonella, Klebsiella pneumoniae, Enterobacter agglomerans, Pantoea dispersa, and Citrobacter cortisone.

4. The use according to claim 1 or 2, characterized in that: The Gram-negative bacteria include those containing colistin resistance genes mcr- 1 Gram-negative bacteria.

5. The use according to claim 4, characterized in that: Contains colistin resistance gene mcr-1 The Gram-negative bacteria include at least one of Escherichia coli, Salmonella, and Klebsiella pneumoniae.

6. An antibacterial drug for Gram-negative bacteria, characterized in that: The antibacterial drug includes colistin and colistin adjuvant, wherein the colistin adjuvant is an antithrombotic small molecule compound BPTU, wherein the antithrombotic small molecule compound BPTU includes a compound as shown in Formula I: Formula I.

7. The antibacterial drug according to claim 6, characterized in that: The colistin includes at least one of polymyxin B and polymyxin E.

8. The antibacterial drug according to claim 6, characterized in that: The drug also includes pharmaceutically acceptable excipients.

9. The antibacterial drug according to claim 8, characterized in that: The auxiliary materials include at least one of a pH regulator, an osmotic pressure regulator, an antibacterial agent, a viscosity regulator, an antioxidant, a penetration enhancer, a diluent, a filler, an adhesive, a wetting agent, an absorption enhancer, a surfactant, a lubricant and a stabilizer.

10. The antibacterial drug according to claim 6, characterized in that: The drugs also include compounds that disrupt bacterial cell membranes.

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