Application of the antithrombotic small molecule compound BPTU as a colistin adjuvant for synergistic anti-Gram-negative bacteria

Abstract

The present application relates to the field of biomedicine technology, and in particular to the use of an antithrombotic small molecule compound BPTU (CAS No. 870544-59-5) as a colistin adjuvant for synergistic anti-Gram-negative bacteria, wherein the antithrombotic small molecule compound BPTU comprises a compound as shown in Formula I; the use of the antithrombotic small molecule compound BPTU as a colistin adjuvant for synergistic anti-Gram-negative bacteria, BPTU and colistin have a synergistic antibacterial effect on Gram-negative bacteria, the outer membrane of Gram-negative bacteria can act as a drug barrier, and colistin has membrane-breaking activity, and colistin breaks the barrier effect of the cell membrane to assist BPTU in entering the cell to exert its 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.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and in particular relates to the application of the anti-thrombotic small molecule compound BPTU as a colistin adjuvant for synergistic anti-Gram-negative bacteria. Background Art

[0002] The 1950s marked the "golden age" of antibiotic discovery, with the discovery of antibiotics such as erythromycin, vancomycin, and metronidazole for treating a wide range of infections. However, few new antibiotics have been discovered since then. The development of entirely new antibiotics is difficult, costly, and time-consuming. Therefore, developing potentiators (adjuvants) for existing antibiotics has become a key issue in combating bacterial resistance.

[0003] The increasing use of polymyxins, coupled with plasmid-borne colistin resistance, is severely undermining the last line of defense against multidrug-resistant Gram-negative pathogens and heralding the emergence of truly pan-drug-resistant infections. Colistin resistance is typically conferred through covalent modification of lipid A with cationic residues such as phosphoethanolamine, mediated by MCR-1, which reduces the affinity of polymyxins for lipopolysaccharides. Therefore, new strategies are needed to address the rapidly diminishing treatment options for Gram-negative infections.

[0004] The antithrombotic small molecule compound BPTU was initially reported to reduce platelet aggregation, thus possessing antithrombotic properties. Based on its potent biological activity and safety, scientists have conducted in-depth research on its targets in mammals. Studies have revealed that BPTU is a non-nucleotide constitutive antagonist of the P2Y1 purinergic receptor, the first member of the P2Y receptor family to be discovered and cloned. The P2Y1 purinergic receptor is a G protein-coupled receptor whose primary endogenous agonists are ADP and ATP. Currently, there are no reports of BPTU's antibacterial activity against Gram-negative bacteria. Given current drug use strategies, discovering a wider range of potentiators for existing antibiotics is crucial for its clinical application. Summary of the Invention

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

[0006] To achieve the above application objectives, the technical solutions adopted in this application are as follows:

[0007] In a first aspect, the present application provides an antithrombotic small molecule compound BPTU for use as a colistin adjuvant for synergistic anti-Gram-negative bacteria, wherein the antithrombotic small molecule compound BPTU comprises a compound as shown in Formula I:

[0008]

[0009] Formula I.

[0010] In some embodiments, the antithrombotic small molecule compound BPTU needs to be used together with colistin to achieve an antibacterial effect against Gram-negative bacteria.

[0011] In some embodiments, the Gram-negative bacteria include at least one of Escherichia coli, Salmonella, Klebsiella pneumoniae, Enterobacter aegypti, Pantoea dispersa, and Citrobacter cohnii.

[0012] In some embodiments, Gram-negative bacteria include those containing colistin resistance genes mcr-1 of Gram-negative bacteria.

[0013] In some embodiments, the colistin resistance gene mcr-1 The Gram-negative bacteria include at least one of Escherichia coli, Salmonella, and Klebsiella pneumoniae.

[0014] In a second aspect, the present application provides an antibacterial drug for Gram-negative bacteria, the antibacterial drug comprising colistin and a colistin adjuvant, wherein the colistin adjuvant is an antithrombotic small molecule compound BPTU, wherein the antithrombotic small molecule compound BPTU comprises a compound as shown in Formula I:

[0015]

[0016] Formula I.

[0017] In some embodiments, the colistin comprises at least one of polymyxin B and polymyxin E.

[0018] In some embodiments, the medicament further comprises a pharmaceutically acceptable excipient.

[0019] In some embodiments, the excipients 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, a binder, a wetting agent, an absorption enhancer, a surfactant, a lubricant, and a stabilizer.

[0020] In some embodiments, the dosage form of the drug includes any one of an injection, a gel preparation, an ointment, and a liquid preparation.

[0021] In some embodiments, the drug also includes compounds that disrupt bacterial cell membranes.

[0022] In the first aspect of the present application, an antithrombotic small molecule compound BPTU is used as a colistin adjuvant for synergistic anti-Gram-negative bacteria. BPTU and colistin have a synergistic antibacterial effect on 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 effect of the cell membrane and assists BPTU in entering the cell to exert its 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.

[0023] The second aspect of the present application provides an antibacterial drug for Gram-negative bacteria, which includes an antithrombotic small molecule compound BPTU and colistin. The combined use of the two can block the survival and spread of colistin-resistant bacteria, becoming a new antibacterial and therapeutic option, and providing new ideas and approaches for the treatment of colistin-resistant Gram-negative bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0025] Figure 1 The CRISPRi screening of Klebsiella pneumoniae under the drug pressure of BPTU provided in the embodiment of the present application screened out the genetic factors of Klebsiella pneumoniae resistance to BPTU yejL and yejM , Phenotypic verification of the outer membrane permeability and sensitivity to the drug BPTU after knocking down these strains, and the results of bacterial morphology observation;

[0026] Figure 2 The results provided in the examples of the present application show that the outer membrane permeability of colistin-resistant Escherichia coli increases at a sub-inhibitory concentration of colistin (CS) and that BPTU reduces HADA binding to the cell wall at a sub-inhibitory concentration of colistin (CS);

[0027] Figure 3 This is a graph showing the synergistic antibacterial activity of BPTU and colistin combined against Escherichia coli ATCC25922, as provided in the examples of the present application;

[0028] Figure 4 The BPTU and colistin combination provided in the embodiment of the present application is a drug containing colistin resistance gene mcr-1 The synergistic antibacterial activity results of Escherichia coli CSZ4;

[0029] Figure 5 This is a graph showing the synergistic antibacterial activity of BPTU and colistin combined against Salmonella ATCC14028, as provided in the examples of the present application;

[0030] Figure 6 The BPTU and colistin combination provided in the embodiment of the present application is a drug containing colistin resistance gene mcr-1 The synergistic antibacterial activity results of Salmonella;

[0031] Figure 7 This is a graph showing the synergistic antibacterial activity of BPTU combined with colistin against Klebsiella pneumoniae CMG1-2, as provided in the examples of the present application;

[0032] Figure 8 This is a graph showing the synergistic antibacterial activity of BPTU and colistin combined against Enterobacter aegypti 1383 provided in the examples of the present application;

[0033] Figure 9 This is a graph showing the synergistic antibacterial activity of BPTU and colistin combined against Citrobacter cohnii 1232 provided in the examples of the present application;

[0034] Figure 10 This is a graph showing the synergistic antibacterial activity of the combination of BPTU and colistin against Pantoea dispersa 1488 provided in the examples of the present application;

[0035] Figure 11 This is a graph showing the sterilization curve results of the combined use of BPTU and colistin provided in the examples of the present application against colistin-resistant Escherichia coli, Klebsiella pneumoniae, and Salmonella;

[0036] Figure 12 The BPTU provided in the embodiment of the present application and the combination of colistin are used to treat colistin-resistant Escherichia coli. E. coli CSZ4( mcr-1 ) in vivo animal experiments. DETAILED DESCRIPTION

[0037] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0038] In this application, the term "and / or" describes the relationship between associated objects, indicating that three possible relationships exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0039] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items. For example, "at least one of a, b, or c" or "at least one of a, b and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can each be single or plural.

[0040] It should be understood that in the various embodiments of the present application, the size of the serial 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 on the implementation process of the embodiments of the present application.

[0041] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.

[0042] The weights of the relevant components mentioned in the examples of this application may not only refer to the specific content of each component, but also represent the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally increased or decreased according to the examples of this application, it is within the scope disclosed in the examples of this application. Specifically, the mass in the examples of this application may be a mass unit known in the chemical industry, such as μg, mg, g, kg, etc.

[0043] The terms "first" and "second" are used solely for descriptive purposes, to distinguish objects, such as substances, from one another. They should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the technical features being referred to. For example, a first XX could also be referred to as a second XX, and similarly, a second XX could also be referred to as a first XX, without departing from the scope of the embodiments of this application. Thus, features defined as "first" or "second" may explicitly or implicitly include one or more of such features.

[0044] The term "CS" is an abbreviation of "Colistin", which means colistin.

[0045] the term" E. coli "for" Escherichia coli ” is the abbreviation for Escherichia coli.

[0046] the term" Sm "for" Salmonella”, which means Salmonella.

[0047] the term" Kp "for" Klebsiella pneumoniae ”, indicating Klebsiella pneumoniae.

[0048] The term "FICI" stands for "Fractional Inhibitory Concentration Index", which means fractional inhibitory concentration index.

[0049] The term "HADA" stands for "7-hydroxycoμMarin-amino-D-alanine," a fluorescent D-alanine derivative widely used to study bacterial cell wall synthesis and dynamics. Its full name is 7-hydroxycoμMarin-3-carboxylic acid–D-alanine. Its structure contains a blue-emitting 7-hydroxycoumarin fluorescent group chemically linked to D-alanine (D-Ala).

[0050] The term "NPN" stands for "N-phenyl-1-naphthylamine", which is a classic fluorescent probe widely used to detect changes in the outer membrane permeability of Gram-negative bacteria.

[0051] "DAPI," short for "4',6-diamidino-2-phenylindole," is a small molecule dye that penetrates cell membranes (especially in fixed or permeabilized cells) and specifically binds to DNA. It particularly readily binds to AT base pairs in DNA and is therefore primarily used to stain nuclear or bacterial nucleic acids.

[0052] The term "Nile Red" stands for "9-diethylamino-5H-benzo[α]phenoxazine-5-one", which is a hydrophobic fluorescent dye that can specifically insert into lipid environments.

[0053] At present, the situation of Gram-negative bacterial infection faced by humans is becoming increasingly severe, especially the spread of drug-resistant strains has increased significantly. These pathogens include those producing extended-spectrum β-lactamases (ESBL) and carbapenem-resistant bacteria (CRE), which are common in urinary tract infections, pneumonia and sepsis. Drug-resistant Gram-negative bacteria are significantly resistant to conventional antibiotics (such as cephalosporins and carbapenems), making treatment more complicated. Colistin (also known as polymyxin E) is a "last line of defense" antibiotic used to treat multidrug-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. Drug resistance is mainly generated through two mechanisms: one is chromosomal mutations that lead to changes in the structure of lipopolysaccharide (LPS), and the other is through plasmid transmission. mcr Genes have been discovered mcr-1 to mcr-10 In response to colistin resistance, various combination therapy strategies are being explored, including the use of polymyxins in combination with other antibiotics, such as sulfamethoxazole / polymyxin and ceftazidime / avibactam. Novel antibiotics, such as cefuroxime and sulfamethoxazole-polymyxin combinations, are also under development, although clinical data remain limited.

[0054] Based on this, the examples of the present application use BPTU as an adjuvant for colistin, and through combined use, the effectiveness of colistin against Gram-negative bacteria is improved, thereby effectively improving the therapeutic effect of colistin against colistin-resistant bacteria, providing new ideas and approaches for the treatment of Gram-negative bacterial infections.

[0055] In a first aspect, an embodiment of the present application provides an antithrombotic small molecule compound BPTU for use as a colistin adjuvant for synergistic anti-Gram-negative bacteria, wherein the antithrombotic small molecule compound BPTU comprises a compound as shown in Formula I:

[0056]

[0057] Formula I.

[0058] In a first aspect of the embodiments of the present application, an antithrombotic small molecule compound BPTU is provided as a colistin adjuvant for synergistic anti-Gram-negative bacteria. BPTU and colistin have a synergistic antibacterial effect on 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 effect of the outer membrane of the cell and assists BPTU in entering the cell to exert its 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.

[0059] In some embodiments, the antithrombotic small molecule compound BPTU is used in conjunction with colistin to achieve antibacterial activity against Gram-negative bacteria. Experimental analysis in the present examples demonstrates that the combined use of BPTU and colistin, at relatively low drug concentrations, can achieve synergistic anti-Gram-negative activity. This provides a new approach for the treatment of Gram-negative bacteria.

[0060] In some embodiments, the Gram-negative bacteria include at least one of Escherichia coli, Salmonella, Klebsiella pneumoniae, Enterobacter aegypti, Pantoea dispersa, and Citrobacter cohnii.

[0061] In some embodiments, Gram-negative bacteria include those containing colistin resistance genes mcr-1 of Gram-negative bacteria.

[0062] In some embodiments, the colistin resistance gene mcr-1 The Gram-negative bacteria include at least one of Escherichia coli, Salmonella, and Klebsiella pneumoniae. Furthermore, when BPTU is used in combination with colistin, at a lower drug concentration of both drugs, that is, at a sub-inhibitory concentration of colistin, the outer membrane permeability of colistin-resistant bacteria can be improved. On this basis, BPTU can cross the barrier of the strain's outer membrane and enter the cell to bind to peptidoglycan, preventing the bacteria from forming cell walls, thereby achieving a bactericidal effect. Therefore, synergistic resistance to bacteria containing colistin resistance genes can be achieved. mcr-1 coli, Klebsiella pneumoniae, and Salmonella. Therefore, this invention provides an effective strategy for treating infections caused by the above strains containing colistin resistance genes.

[0063] In addition, when BPTU is used in combination with colistin, at lower drug concentrations of both drugs, it can have a good therapeutic effect on infection in mice. Therefore, it further proves that the combination of BPTU and colistin has broad application prospects in clinical practice.

[0064] A second aspect of the embodiments of the present application provides an antibacterial drug for Gram-negative bacteria, the antibacterial drug comprising colistin and a colistin adjuvant, wherein the colistin adjuvant is an antithrombotic small molecule compound BPTU, wherein the antithrombotic small molecule compound BPTU comprises a compound as shown in Formula I:

[0065]

[0066] Formula I.

[0067] The second aspect of the embodiment of the present application provides an antibacterial drug for Gram-negative bacteria, which includes the anti-thrombotic small molecule compound BPTU and colistin. The combined use of the two can block the survival and spread of colistin-resistant bacteria, becoming a new antibacterial and therapeutic scheme, and providing new ideas and approaches for the treatment of colistin-resistant Gram-negative bacterial infections.

[0068] In some embodiments, the colistin comprises at least one of polymyxin B and polymyxin E.

[0069] In some embodiments, the medicament further comprises a pharmaceutically acceptable excipient.

[0070] In some embodiments, the excipients 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, a binder, a wetting agent, an absorption enhancer, a surfactant, a lubricant, and a stabilizer.

[0071] In some embodiments, the dosage form of the drug includes any one of an injection, a gel preparation, an ointment, and a liquid preparation.

[0072] In some embodiments, the drug also includes compounds that disrupt bacterial cell membranes.

[0073] The following describes the details in conjunction with specific embodiments.

[0074] Example 1

[0075] Screening of small molecule compound libraries for compounds that can produce synergistic anti-Gram-negative bacteria effects with colistin.

[0076] From targetMol ( https: / / www.targetmol.cn / search?keyword=BPTU ) Purchase the small molecule compound library with product number: T4132. The BPTU in the compound library is combined with colistin for drug sensitivity test. The process is as follows:

[0077] First, Escherichia coli ATCC25922, Salmonella ATCC14028, Klebsiella pneumoniae CMG1-2, Enterobacter agglomerans, Pantoea dispersae, and Citrobacter cohnii preserved in our laboratory were inoculated into LB test tube broth and cultured at 37°C and 180 rpm for 4 h. The culture solution was dipped with an inoculating loop and a single colony was streaked on LB agar medium, which was then cultured in a constant temperature incubator at 37°C overnight.

[0078] 1.1 Determination of strain MIC

[0079] The experiment was conducted according to the CLSI operating standards. Escherichia coli ATCC25922, Salmonella ATCC14028, Klebsiella pneumoniae CMG1-2, Enterobacter agglomerans, Pantoea dispersa, and Citrobacter cohnii were inoculated into fresh MH test tube broth and cultured at 37°C and 180 rpm for 4 h until the bacterial solution concentration reached 0.5 McFarland turbidity. The solution was diluted 100 times to 1 × 10 6 CFU / ml bacterial volume was used for standby use. Escherichia coli ATCC25922, Salmonella ATCC14028, Klebsiella pneumoniae CMG1-2, Enterobacter aegypti, Pantoea dispersa, and Citrobacter cohnii were inoculated into fresh MH test tube broth and cultured at 37°C and 200 rpm for 6-8 h until the bacterial solution concentration reached 0.5 McFarland turbidity. The solution was diluted 100-fold to 1×10 6 The CFU / ml bacterial count is set aside. The drug preparation and dilution methods are shown in Table 1, and the antibiotic susceptibility results are shown in Table 2.

[0080] Agar dilution method: Add 1 ml of the test drug and its corresponding diluent (Table 1) to a sterile blank plate. Then, dilute the solution to various drug concentrations using a two-fold dilution method, and record the drug concentrations on each plate. After the autoclaved MH agar cools, use a pipette to transfer 19 ml of MH agar to each plate. After the agar solidifies, use an inoculator to inoculate the diluted bacterial solution onto MH agar plates containing various drug concentrations and a blank control medium. After the bacterial solution has dried at room temperature, incubate at 37°C for 18 hours, and record the MIC values ​​of the different antibiotics.

[0081] Broth dilution method: Add 180 μL of MH broth to the first column of a 96-well plate, and 100 ml of MH broth to each of the remaining wells. Then, add 20 μL of 5120 mg / L antibiotic to each well in the first column. After thorough mixing, perform a two-fold serial dilution from left to right. Finally, add 100 μL of the diluted bacterial solution to each well and incubate at 37°C for 18–22 hours. Observe and interpret the results. Interpret the MICs for E. coli ATCC 25922 according to the CLSI MIC quality control range. If the results are consistent with the CLSI standard, the MIC results for this experiment are reliable.

[0082] Table 1

[0083]

[0084] Table 2

[0085]

[0086] Table 2 refers to the M100 standard, 32nd edition (2022), published by the Clinical and Laboratory Standards Institute (CLSI).

[0087] 1.2 Checkerboard test:

[0088] Using colistin as drug A and a small molecule compound as drug B, add 50 μL of fresh MH broth to columns 1 to 8 of a 96-well plate, dilute drug A to 16 times its MIC value (because drug A is diluted in series inside the plate, the highest final concentration of drug A is 2 times the MIC), and dilute drug B to 8 times its MIC value (because 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 with a 16 MIC value to wells 1 to 8 of row A, use an 8-channel gun with the range adjusted to 50 μL, dilute from row A to row G, and discard the gun tip; when drug B is diluted to 8 MIC values, dilute it in series in 6 gradients in a clean plate, with the concentrations being 8 MIC values, 4 MIC values, 2 MIC values, 1 MIC value, 1 / 2 MIC value, 1 / 4 MIC value, 1 / 8 MIC values ​​were calculated by adding 7 gradients to rows A to H in the first column, rows A to H in the second column, and rows A to H in the seventh column, and adding 50 μL of MH broth to rows A to H in the eighth column. ATCC 25922 grown to the logarithmic phase was diluted 100-fold in a blank plate to obtain a concentration of about 10 6 CFU / ml, add 100 μL to each well of columns 1-8; then incubate in a 37°C incubator for 18-22 hours and determine the results. Each experiment was repeated three times.

[0089] Result determination: In the laboratory, the basis for judging the combined drug sensitivity test is to calculate the fractional inhibitory concentration index (FICI):

[0090] FICI index = MIC of drug A in combination / MIC of drug A alone + MIC of drug B in combination / MIC of drug B alone.

[0091] Table 3

[0092]

[0093] 1.3 Effect of BPTU combined with colistin on bacteria containing colistin-resistant genes mcr-1 Bactericidal experiment of strains

[0094] 1. High pressure: 100 ml LB broth, MHA plate, 200 ml physiological saline, several 2 ml EP tubes, 100 ml pure water, filter membrane, several 1 ml and 200 μL pipette tips, and several 10 ml EP tubes.

[0095] 2. Bacterial culture: At 9 pm, the bacteria containing the colistin resistance gene were cultured. mcr-1 coli, Klebsiella pneumoniae, and Salmonella were added to LB test tube broth and allowed to stand at 37°C overnight. The next morning, the culture was placed in a 37°C shaker for 30 min, the OD value was measured, and the bacterial solution was diluted to 1×10 6 CFU / ml.

[0096] 3. According to the experimental results of combined drug use, 1×10 6 The drugs were diluted to the corresponding concentrations (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).

[0097] 4. After mixing, incubate 1 ml of the solution in a 5 ml flow cytometer tube. This will be the start time at 0 h. Then, perform serial dilutions and count the samples at 4, 8, 12, and 24 h.

[0098] 1.4 CRISPRi screening of genes associated with BPTU sensitivity in Klebsiella pneumoniae.

[0099] The Klebsiella pneumoniae CRISPRi library was pre-induced for 7 generations and 14 generations. The library stored at -80°C was inoculated into 4 ml of LB broth at a dilution of 1:100 and placed in a shaker at 37°C and 250 rpm for activation (OD 600 = 0.6), then transferred into 4 ml containing 1 mM IPTG at 1:100 and placed in a shaker at 37 °C and 250 rpm for 7 generations of pre-induction (OD of the induced strain was 0.6). 600 = about 0.6). The 7th generation pre-induced library was again inoculated with 4 ml containing 1 mM IPTG at a dilution of 1:100 for 14 generations of pre-induction (OD of the induced strain was 600 = approximately 0.6).

[0100] Screening with a 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 groups and treatment conditions, as well as the extracted genome concentrations after treatment, are shown in the following table:

[0101] Table 4

[0102]

[0103] Screening at 14 generations: 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 groups and treatment conditions, as well as the extracted genome concentration after treatment, are shown in the following table:

[0104] Table 5

[0105]

[0106] Using the above-extracted genome as a template, the sgRNA sample pool sequenced by illμMina was amplified using a high-fidelity enzyme. The primer sequences used for PCR and the PCR amplification procedure are as follows:

[0107] Table 6: Primer sequences used

[0108]

[0109] Table 7: PCR reaction system

[0110]

[0111] Table 8: Reaction procedures for the above PCR

[0112]

[0113] 1.5 NPN knockdown strain determination yejM and yejL Outer membrane permeability after genes.

[0114] Pick CRISPRi target cells from LB agar plates yejM 、 yejL, gfp Single colonies of the gene (Nontargeting) were inoculated into 1 ml LB broth and induced with 0.1 mM IPTG. yejM gene, induced with 0.05 mM IPTG yejL Each group of three parallel genes was shaken at 37°C and 220 rpm until OD 600 =0.3, the supernatant was removed by centrifugation. The culture was then washed with 5 μM HEPES (pH 7.0 + 5 μM glucose), suspended, and repeated twice. The washed culture was then diluted 1:10 into HEPES (pH 7.0 + 5 μM glucose), and finally, the dye NPN was added to a final concentration of 10 μM. 200 μl of probe-labeled bacterial cells were added to a 96-well microplate reader. After incubation for 30 minutes, fluorescence was measured in an Infinite M200 Microplate reader (Tecan) at an excitation wavelength of 350 nm and an emission wavelength of 420 nm.

[0115] 1.6 NPN determination of strains treated with subinhibitory concentrations of colistin E. coli Outer membrane permeabilization by CSZ4.

[0116] Store at -80 degrees Celsius E. coliCSZ4 was inoculated into 3 ml of fresh LB broth at a dilution ratio of 1:100 and cultured in a shaker at 37°C and 250 rpm until the strain reached an OD 600 = about 0.6. Then, the strain was inoculated into LB broth containing different colistin concentrations at a ratio of 1:100, namely 0×MIC, 1 / 8×MIC, 1 / 4×MIC, 1 / 2×MIC, and 1×MIC, with 3 replicates per group. The above groups were cultured in a shaker at 37°C and 250 rpm until the strain grew to OD 600 =0.3, and the supernatant was removed by centrifugation. The culture was then washed with 5 μM HEPES (pH 7.0 + 5 μM glucose), suspended, and repeated twice. The washed culture was then diluted 1:10 into HEPES (pH 7.0 + 5 μM glucose), and finally, the dye NPN was added to a final concentration of 10 μM. 200 μl of probe-labeled bacterial cells were added to a 96-well microplate reader. After incubation for 30 minutes, fluorescence was measured on an Infinite M200 Microplate reader (Tecan) at an excitation wavelength of 350 nm and an emission wavelength of 420 nm.

[0117] 1.7 Microscopic observation of CRISPRi knockdown strains yejM and yejL Genes followed by strain morphology.

[0118] CRISPRi knockdown strains yejM and yejL Transfer the overnight cultured strain to fresh medium, add IPTG and NileRed for induction and staining until the bacterial count reaches OD 600 The value is about 0.6; the above culture medium was centrifuged, the supernatant was removed, and 1 ml of 4% PFA fixative was added at the same time, and the solution was protected from light for 1 h; after fixation, the supernatant was removed by centrifugation (2400xg, 4 min), and the cells were resuspended with an equal volume of prepared QS (100mMTris) (to remove residual PFA); 2% Agarose was prepared in a prefabricated mold for sample loading and observation, and the corresponding dye was added during the preparation; the bacterial solution after QS resuspending was dropped onto the gel platform, covered with a coverslip, and the image was collected on the machine; the dye working concentrations were: DAPI 1 μg / ml, Nile Rea 0.2 μg / ml, SYTOX 1:2000.

[0119] 1.8 Determination of CRISPRi knockdown strains yejM and yejL The sensitivity of the strain to BPTU and vancomycin was evaluated after the gene was expressed.

[0120] CRISPRi knockdown strains yejM and yejLTake out the bacteria from the -80℃ storage library and inoculate into fresh LB broth at a ratio of 1:100, shake at 37℃ until the OD 600 The two strains were inoculated into new LB broth at a dilution ratio of 1:100, and the mixture was set to add IPTG or not and continued to shake until the OD 600 It is about 0.6, and then it is diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Gradient; take 10 μL of the above bacterial droplets and add IPTG - 、IPTG + 、IPTG - +BPTU + / Van + 、IPTG + +BPTU + / Van + The LB agar plates were plated with BPTU at a concentration of 5 μg / ml and vancomycin at a concentration of 5 μg / ml. The plates were incubated at 37°C overnight.

[0121] Result Analysis

[0122] 1 The antibacterial effect of the small molecule compound BPTU on selected Gram-negative bacteria is shown in Table 9.

[0123] Table 9

[0124]

[0125] It can be seen that the small molecule compound BPTU represented by the provided formula I has no antibacterial effect on Klebsiella pneumoniae CMG1-2, Salmonella typhimurium GP9, Salmonella typhimurium ATCC14028, Escherichia coli CSZ4, Escherichia coli ATCC25922, Enterobacter agglomerans, Pantoea dispersa, and Citrobacter cohnii at a concentration of up to 256 μg / ml.

[0126] 2. In order to explore the genetic basis of natural resistance of Gram-negative bacteria to BPTU on a genome-wide scale. In the present embodiment, a CRISPRi screening of clinical multidrug-resistant Kp (Klebsiella pneumoniae) strains under the pressure of BPTU was performed. This screening uses a CRISPRi library covering the entire genome, which can induce knockdown of almost all genes in Kp, thereby quantifying the chemical-genetic interactions of essential and non-essential genes and providing a global overview of gene-drug interactions in Kp. After the library was induced in advance, the present embodiment used 16 μg / ml of BPTU to act on the Kp library. For about 3 hours (bacterial solution concentration OD 600 =0.6), genomic DNA was extracted from the culture, amplified with specific primers, and analyzed for sgRNA (single-stranded guide RNA) abundance by deep sequencing. The selected genes were analyzed and identified using 2fastq2 software. In summary, the present invention identified two genes that, after knockdown, caused Kp to become sensitive to BPTU from natural resistance, while no gene knockdown caused Kp to become more resistant to BPTU ( Figure 1 The two sensitizing genes are PCBIOHEG_01623 ( YejM ) and PCBIOHEG_01624 ( yejL Hypothetical protein of unknown function) Figure 1 B). Among them, the inner membrane protein PCBIOHEG_01623 ( YejM ) is a metalloenzyme that regulates LPS biosynthesis ( Figure 1 C). Knockdown was performed under conditions of IPTG concentrations of 100 μM and 50 μM. yejM and yejL , it can be seen that knocking down the two genes under this condition did not affect the normal growth of the strain (such as Figure 1 After knocking down these two genes, the strain's sensitivity to BPTU and vancomycin was significantly increased in a culture medium with a BPTU and vancomycin concentration of 5 μg / ml (e.g. Figure 1 Considering that inhibiting them makes Kp sensitized to BPTU, in order to clarify the mechanism by which PCBIOHEG_01623-PCBIOHEG_01624 proteins mediate BPTU resistance, the present invention uses 1-N-phenylnaphthylamine (1-Naphthylphenylamine NPN) to measure the permeability of the strain outer membrane after knocking down the two sensitizing genes. The results show that after knocking down the two genes, the outer membrane permeability is significantly increased compared with the control group (sgRNA-non-targeting). Figure 1In addition, the morphology of the two genes after knockdown was further observed under a microscope. It can be seen that the control group (sgRNA-non-targeting) did not show morphological changes, while the sgRNA targeting PCBIOHEG_01623 and PCBIOHEG_01624 bacteria changed the normal morphology of the bacteria into a "spherical" shape (such as Figure 1 J and K). In addition, based on the above results, it can be seen that inhibiting the Kp outer membrane can make the strain sensitive to BPTU instead of resistant. In addition, the present invention tested the outer membrane permeability of the strain after treatment with sub-inhibitory concentrations of colistin. The results showed that the outer membrane permeability of the strain was significantly reduced after treatment with sub-inhibitory concentrations of colistin (1 / 2, 1 / 4 MIC). Figure 2 A), at the same time, the present application embodiment tested that BPTU can significantly reduce the ability of HADA to bind to the cell wall under the treatment of sub-inhibitory colistin (1 / 2MIC) ( Figure 2 B), indicating that after colistin changes the cell membrane permeability, BPTU effectively enters the cell and prevents cell wall synthesis. Therefore, this example of the application combines BPTU with the membrane-targeting agent colistin to explore the in vitro and in vivo effects of the combined use of the two.

[0127] 3. The synergistic antibacterial activity of the small molecule compound BPTU combined with colistin against Escherichia coli ATCC25922 is shown in the following table. Figure 3 As shown in the figure, when colistin is used alone, its MIC is 2.0 μg / ml. However, when used in combination with BPTU, the strain's resistance to colistin drops to 0.25 μg / ml at a BPTU concentration of 1.0 μg / ml. According to the calculation formula for the combined use, the FICI for the combined use of the two drugs is 0.1289, which is less than 0.5. Therefore, the two drugs act synergistically.

[0128] The combination of small molecule compound BPTU and colistin can inhibit the expression of colistin resistance gene mcr-1 Escherichia coli E. coli CSZ4( mcr-1 ) synergistic antibacterial activity results are as follows Figure 4 As shown in the figure, when colistin was used alone, the MIC was 4.0 μg / ml, indicating colistin resistance. However, when used in combination with BPTU, at a BPTU concentration of 2 μg / ml, the strain's resistance to colistin dropped to 0.5 μg / ml, becoming susceptible to colistin. According to the formula for combined use, the FICI for the combined use of the two drugs was 0.1328, which is less than 0.5, indicating a synergistic effect.

[0129] The synergistic antibacterial activity of the small molecule compound BPTU combined with colistin against Salmonella ATCC14028 Figure 5As shown in the figure, when colistin is used alone, its MIC is 1.0 μg / ml. However, when used in combination with BPTU, the strain's resistance to colistin drops to 0.125 μg / ml at a BPTU concentration of 1.0 μg / ml. According to the calculation formula for the combined use, the FICI for the combined use of the two drugs is 0.1289, which is less than 0.5. Therefore, the two drugs act synergistically.

[0130] The synergistic antibacterial activity of the small molecule compound BPTU combined with colistin against Salmonella containing the colistin resistance gene mcr-1 is shown in Figure 2. Figure 6 As shown in the figure, when colistin was used alone, the MIC was 4.0 μg / ml, indicating colistin resistance. However, when used in combination with BPTU, at a BPTU concentration of 2.0 μg / ml, the strain's resistance to colistin dropped to 0.5 μg / ml, becoming susceptible to colistin. According to the formula for combined use, the FICI for the combined use of the two drugs was 0.2578, which is less than 0.5, indicating a synergistic effect.

[0131] The synergistic antibacterial activity of the small molecule compound BPTU combined with colistin against Klebsiella pneumoniae CMG1-2 Figure 7 As shown in the figure, when colistin was used alone, the MIC was 8.0 μg / ml, indicating colistin resistance. However, when used in combination with BPTU, the strain's resistance to colistin dropped to 1.0 μg / ml at a BPTU concentration of 2.0 μg / ml, becoming susceptible to colistin. According to the formula for the combined use, the FICI for the combined use of the two drugs was 0.2578, which is less than 0.5, indicating a synergistic effect.

[0132] The synergistic antibacterial activity of the small molecule compound BPTU combined with colistin against Enterobacter aegypti 1383 Figure 8 As shown in the figure, when colistin is used alone, its MIC is 8.0 μg / ml. However, when used in combination with BPTU, the strain's resistance to colistin drops to 2.0 μg / ml at a BPTU concentration of 4.0 μg / ml. According to the calculation formula for the combined use, the FICI for the combined use of the two drugs is 0.1406, which is less than 0.5. Therefore, the two drugs act synergistically.

[0133] The synergistic antibacterial activity of the small molecule compound BPTU combined with colistin against Citrobacter cohnii 1232 Figure 9As shown in the figure, when colistin is used alone, its MIC is 0.5 μg / ml. However, when used in combination with BPTU, the strain's resistance to colistin drops to 0.125 μg / ml at a BPTU concentration of 0.25 μg / ml. According to the calculation formula for the combined use, the FICI for the combined use of the two drugs is 0.2509, which is less than 0.5. Therefore, the two drugs act synergistically.

[0134] The synergistic antibacterial activity of the small molecule compound BPTU combined with colistin against Pantoea dispersa 1488 was shown in Figure 2. Figure 10 As shown in the figure, when colistin is used alone, its MIC is 1.0 μg / ml. However, when used in combination with BPTU, the strain's resistance to colistin drops to 0.25 μg / ml at a BPTU concentration of 0.25 μg / ml. According to the calculation formula for the combined use, the FICI for the combined use of the two drugs is 0.2509, which is less than 0.5. Therefore, the two drugs act synergistically.

[0135] The combined use of small molecule compound BPTU and colistin against colistin-resistant Escherichia coli, Klebsiella pneumoniae, and Salmonella is shown in the following figure: Figure 11 The results show that when colistin 2.0 μg / ml or BPTU 8.0 μg / ml was used alone, there was no significant bactericidal effect, but when colistin 2.0 μg / ml was used in combination with BPTU 8.0 μg / ml, a significant bactericidal effect was seen.

[0136] Example 2

[0137] In vivo combination drug studies

[0138] 2.1 Effects of BPTU combined with colistin mcr-1 Evaluation of the efficacy of a positive bacteria skin infection model

[0139] ① Male BaLB / C mice, 6-8 weeks old, weighing 20-25 g.

[0140] ②-3 days: shaving, depilatory cream.

[0141] ③-2 days: Wound test (8 mm in diameter, 6 mm in depth), anesthetic;

[0142] ④-1 day: Bacterial infection, drip on the wound (30 μL 1x10 8 CFU / ml), and one day later, the wound exudate was collected and smeared on an agar plate to see if there were colonies to verify whether the infection model was successful;

[0143] ⑤Day 0: Start treatment.

[0144] Based on the above in vitro combined drug results, the following experimental groups were set up:

[0145] ①Blank group (wound exposed without any treatment);

[0146] ②Solvent DMSO group (100 μL, pH=7.0);

[0147] ③BPTU group (100 μL 2.0 μg / ml);

[0148] ④ Colistin group (100 μL 2.0 μg / ml);

[0149] ⑤BPTU+colistin group (100 μL 2.0 +2.0 μg / ml);

[0150] The drug was dripped into the wound once a day, 50 μL each time for 7 consecutive days.

[0151] index:

[0152] 1. Wound photography: Take photos every day for 7 consecutive days and count the wound healing rate;

[0153] 2: Weight changes: 1-7 days;

[0154] 3: Bacterial count: On the 7th day, the wound tissue was collected and placed in a centrifuge tube containing 1 ml of PBS. The tissue was thoroughly ground and incubated at 37°C for 1 h before plating on a plate.

[0155] Result analysis:

[0156] like Figure 12 As shown, Figure 12 A is BPTU combined with colistin to treat Escherichia coli infection E. coli CSZ4( mcr-1 Schematic diagram of the efficacy of the mouse model with positive results. Figure 12 Figure B is a representative photo of the skin healing process after medication treatment. It can be seen that from the third day to the seventh day after treatment, the wounds in the combination group were smaller than those in the control group, solvent group, colistin monotherapy group, and BPTU monotherapy group. Figure 12 C is the laser speckle pattern of the wound in each group on the 1st to 7th day of treatment (in the black dotted box); it can be seen that the combined drug treatment group. Figure 12 D is the weight change of mice from day 1 to day 7 after treatment; it can be seen that the weight of mice in the combination treatment group began to increase from day 3, and the growth rate was higher than that of the other groups mentioned above. Figure 12E is the wound area calculated using ImageJ software. The data were statistically analyzed based on the lesion size of each treatment group on the 7th day. The black dotted lines represent the 50% and 80% healing rates. It can be seen that the wound healing rate of the combination medication group reached 50% on the 3rd day after treatment and reached 80% on the 6th day of treatment, while the other groups mentioned above did not achieve the same healing rate as the combination medication group within the same treatment time. Figure 12 F is the bacterial load of BPTU in reducing skin wound infection; it can be seen that the mean bacterial load of mice in the combined treatment group was 1×10 4 CFU / ml, while the bacterial loads of the other groups mentioned above were close to 1×10 7 CFU / ml. Calculation of E. coli colonization in wounds on day 7 after infection in the skin model of this application E. coli CFU of CSZ4. P The values ​​were analyzed by one-way ANOVA. Each bar is the mean ± SD of 5 independent biological replicates. P The values ​​are <0.05 and <0.01, indicated by “*” and “**”, respectively.

[0157] In summary, the examples of the present application provide an antithrombotic small molecule compound BPTU as an adjuvant for colistin to synergistically fight Gram-negative bacteria. BPTU and colistin have a synergistic antibacterial effect on 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 effect of the outer membrane of the cell and assists BPTU in entering the cell to exert its 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.

[0158] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. Use of an antithrombotic small molecule compound BPTU as a colistin adjuvant in the preparation of a drug for treating Gram-negative bacteria, wherein: The antithrombotic small molecule compound BPTU includes a compound as shown in Formula I: Formula I; Wherein, the Gram-negative bacteria include those containing colistin resistance genes mcr- 1. At least one of Escherichia coli, Klebsiella pneumoniae, and Salmonella.

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. An antibacterial drug for Gram-negative bacteria, characterized in that: The antibacterial drug includes colistin and a 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; Wherein, the Gram-negative bacteria include those containing colistin resistance genes mcr- 1. At least one of Escherichia coli, Klebsiella pneumoniae, and Salmonella.

4. The antibacterial drug according to claim 3, characterized in that The colistin includes at least one of polymyxin B and polymyxin E.

5. The antibacterial drug according to claim 3, characterized in that The drug also includes pharmaceutically acceptable excipients.

6. The antibacterial drug according to claim 5, 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.

7. The antibacterial drug according to claim 3, characterized in that Such drugs also include compounds that disrupt bacterial cell membranes.

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