Application of cell division protein FtsZ targeted inhibitor A3 in the preparation of drugs against drug-resistant bacteria

Abstract

The present invention provides an application of a cell division protein FtsZ targeted inhibitor in the preparation of an anti-drug resistant bacteria drug, belonging to the technical field of drug research and development. The inhibitor provided by the present invention is compound A3, named (1-methyl-2-((Z)-(3-methylbenzo[d]thiazole-2(3H)-ylidene)methyl)-4-((E)-4-(4-methylpiperazine-1-yl)styryl)quinoline-1-iodide, with a molecular formula of C 32 H 33 IN4S. The compound A3 has a significant inhibitory effect on multiple drug-resistant bacteria, especially vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus, and can significantly increase the sensitivity of methicillin to methicillin-resistant Staphylococcus aureus.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to application of a cell division protein FtsZ targeted inhibitor in the preparation of drugs against drug-resistant bacteria. Background Art

[0002] Bacterial infections are primarily treated with antibiotics in clinical practice. With the widespread use of antibiotics, bacterial resistance is becoming increasingly serious. For example, the emergence of methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Staphylococcus aureus (MDRSA), vancomycin-resistant Enterococci (VRE), and extended-spectrum β-lactamase (ESBLs) and NDM-1 (New Delhi Metallo-β-lactamase 1)-positive strains has greatly challenged clinical medication use, with some resistant strains even facing a situation where no drugs are available. Another concern is the relatively slow development of new antimicrobial drugs.

[0003] Recent studies have shown that FtsZ (filamenting temperature-sensitive protein Z) is an indispensable protein in the bacterial division process. It plays a vital role in the bacterial division and proliferation process. It exists in almost all bacteria and is highly conserved in morphology and not prone to mutation. When its biological activity is disturbed, the growth and reproduction of bacteria will be inhibited [2]. Therefore, the study of antibacterial molecules targeting FtsZ has important reference value for the development of a new generation of antibacterial drugs. At present, there are no reports that 4-(4-methylpiperazin-1-yl)phenylvinyl-quinoline derivatives have the ability to target FtsZ protein, induce bacterial division, and thus exert antibacterial activity. Summary of the Invention

[0004] In response to the above problems, the purpose of the present invention is to provide the use of a cell division protein FtsZ targeted inhibitor A3 in the preparation of anti-resistant bacteria drugs. The present invention provides an FtsZ targeted inhibitor A3, which has a significant inhibitory effect on a variety of drug-resistant bacteria and has important practical significance for the research and development of anti-resistant bacteria drugs.

[0005] The specific scheme adopted in the present invention is:

[0006] In the first aspect, a bacterial fission protein FtsZ targeted inhibitor is provided, wherein the inhibitor is compound A3, named (1-methyl-2-((Z)-(3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)-4-((E)-4-(4-methylpiperazin-1-yl)phenylvinyl)quinoline-1-iodide, with a molecular formula of C 32 H 33IN4S, the structural formula is shown in formula (I):

[0007]

[0008] In a second aspect, the above inhibitors are used in any of the following: (1) in vitro antibacterial activity; (2) preparation of drugs for treating drug-resistant bacteria.

[0009] In a third aspect, an antibacterial drug comprises the above-mentioned inhibitor.

[0010] The antibacterial drugs are drugs against Staphylococcus aureus, Escherichia coli, Enterococcus and Bacillus subtilis.

[0011] The Staphylococcus aureus is methicillin-resistant Staphylococcus aureus.

[0012] The Escherichia coli is drug-resistant Escherichia coli.

[0013] The enterococci are vancomycin-resistant enterococci.

[0014] The antibacterial drug also contains pharmaceutically acceptable excipients or other compatible drugs.

[0015] The pharmaceutically acceptable excipients are pharmaceutical excipients, including solvents, disintegrants, flavoring agents, preservatives, colorants or adhesives.

[0016] The dosage form of the antibacterial drug is injection, tablet, pill, capsule, suspension or emulsion.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0018] (1) The targeted FtsZ protein inhibitor A3 of the present invention has a significant inhibitory effect on multiple drug-resistant bacteria, especially vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus;

[0019] (2) The targeted FtsZ protein inhibitor A3 of the present invention can significantly increase the sensitivity of methicillin to methicillin-resistant Staphylococcus aureus. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Graph (40×) showing the effects of the blank control (Panel A) and the FtsZ protein inhibitor A3 (Panel B) on the growth morphology of Bacillus subtilis in Example 3.

[0021] Figure 2 This is a graph showing the effect of the FtsZ protein inhibitor A3 on the dynamic aggregation of the FtsZ protein in Example 4. DETAILED DESCRIPTION

[0022] The present invention provides an FtsZ protein inhibitor, which is compound A3 (1-methyl-2-((Z)-(3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)-4-((E)-4-(4-methylpiperazin-1-yl)styryl)quinolin-1-ium iodide, 1-methyl-2-((Z)-(3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)-4-((E)-4-(4-methylpiperazin-1-yl)styryl)quinolin-1-ium iodide), whose molecular formula is C 32 H 33 IN4S, the structural formula is shown in formula (I):

[0023] The above-mentioned targeted FtsZ protein inhibitor A3 can be used to prepare antibacterial drugs.

[0024] The antibacterial drug is a drug against Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus.

[0025] The antibacterial drug is a drug against Escherichia coli, including drug-resistant Escherichia coli.

[0026] The antibacterial drug is an anti-enterococcal drug, including vancomycin-resistant enterococci.

[0027] The antibacterial drug is a drug against Bacillus subtilis.

[0028] The antibacterial drug is a drug that can increase the sensitivity of methicillin to methicillin-resistant Staphylococcus aureus.

[0029] The antibacterial drug also contains pharmaceutically acceptable excipients or other compatible drugs.

[0030] The pharmaceutically acceptable excipients mentioned herein refer to conventional pharmaceutical excipients, such as solvents, disintegrants, flavoring agents, preservatives, colorants and adhesives.

[0031] The dosage form of the anti-drug-resistant bacteria drug is preferably an injection, tablet, pill, capsule, suspension or emulsion.

[0032] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0033] Example 1 Test of antibacterial activity

[0034] In this example, the minimum inhibitory concentration (MIC) (μg / mL) of the FtsZ protein inhibitor A3 was determined using the broth microdilution method. The minimum inhibitory concentration (MIC) of the test compound was determined according to the broth microdilution procedure described in the Clinical and Laboratory Standards Institute (CLSI) guidelines.

[0035] The implementation steps are as follows:

[0036] (1) Preparation of culture medium and antimicrobial drug stock solution: The prepared MH broth culture medium was sterilized by autoclaving at 121°C for 30 min and then cooled; the test compound was dissolved in DMSO to prepare a 38.4 mg / mL stock solution, and then sterilized by filtration using a 0.22 μm filter membrane.

[0037] (2) Activation and expansion of the test bacteria: Spread the frozen test bacteria on TSB agar plates and activate and culture at 37°C for 24 h; inoculate the single colony obtained from the activation culture into 5 mL of TSB medium and culture at 37°C with shaking overnight.

[0038] (3) Seed plate: Measure the OD value at a wavelength of 600 nm using a microplate reader to calculate the bacterial concentration. Dilute the bacterial solution with MH broth to a concentration of approximately 5 × 10 5 CFU / mL, add blank culture medium to the outermost well of the 96-well plate, and add 100 μL of bacterial solution to each of the remaining wells for later use.

[0039] (4) Drug addition: The blank culture medium at the outermost edge of the 96-well plate was used as a blank control. 96 μL of bacterial solution was added to each well of the second column, and then 4 μL of the compound stock solution prepared in step 1 was added. After thorough mixing, 100 μL of the mixed solution was pipetted and added to the third column. After thorough mixing again, 100 μL of the mixed solution was pipetted and added to the fourth column. When the process was repeated to the 11th column, 100 μL of the mixed solution was pipetted and discarded. That is, the drug was added using the half-dilution method. The final drug concentrations in each well of the second to 11th columns were 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL, respectively. DMSO was used as the solvent control, and ampicillin (AMP), vancomycin (VAN), methicillin (MET), and rifampicin (RIF) were used as positive controls.

[0040] (5) Incubation: Place the 96-well plate in a 37°C constant temperature incubator and incubate for 24 h.

[0041] (6) Result judgment: The blank control wells showed no obvious bacterial growth and were clear. The lowest drug concentration at which the wells were completely clear and transparent was taken as the MIC value, i.e., visible clarity indicated 90% inhibition of bacterial growth.

[0042] (7) Test bacteria: The bacteria used in the microbroth dilution method include Staphylococcus aureus ATCC29213, methicillin-resistant Staphylococcus aureus ATCC BAA-41, methicillin-resistant Staphylococcus aureus ATCC 43300, multidrug-resistant Staphylococcus aureus ATCC BAA-44, vancomycin-resistant Staphylococcus aureus VISA, Staphylococcus epidermidis ATCC 12228, methicillin-resistant Staphylococcus epidermidis MRSE, Bacillus subtilis 168, Escherichia coli ATCC 25922, multidrug-resistant Escherichia coli BAA-2469, Enterococcus faecium ATCC 49624, vancomycin-resistant Enterococcus faecium ATCC 700221.

[0043] (8) The test results are shown in Table 1. The test compounds can inhibit the reproduction of multiple drug-resistant bacteria in vitro and can be used to prepare antibiotic drugs against drug-resistant bacteria.

[0044] Table 1 MIC values ​​of the test compounds against the test bacteria (μg / mL)

[0045]

[0046] Example 2 FtsZ protein inhibitor A3 increases methicillin sensitivity to methicillin-resistant Staphylococcus aureus (MRSA)

[0047] The implementation steps are as follows:

[0048] (1) The compound and methicillin were used in combination to fight against methicillin-resistant Staphylococcus aureus (MRSA) using the checkerboard assay, based on the determination of the MICs of the compound and methicillin.

[0049] (2) Methicillin was prepared into a 50 mg / mL stock solution using DMSO and filtered for sterilization.

[0050] (3) Recovery and expansion culture: refer to the minimum inhibitory concentration (MIC) experiment.

[0051] (4) The distribution of drug concentrations is shown in Table 2 below (the values ​​outside the brackets are compound MIC multiples, and the values ​​inside the brackets are methicillin MIC multiples).

[0052] Table 2 Distribution of drug concentration

[0053] 1 / 32(1) 1 / 32(1 / 2) 1 / 32(1 / 4) 1 / 32(1 / 8) 1 / 32(1 / 16) 1 / 32(1 / 32) 1 / 16(1) 1 / 16(1 / 2) 1 / 16(1 / 4) 1 / 16(1 / 8) 1 / 16(1 / 16) 1 / 16(1 / 32) 1 / 8(1) 1 / 8(1 / 2) 1 / 8(1 / 4) 1 / 8(1 / 8) 1 / 8(1 / 16) 1 / 8(1 / 32) 1 / 4(1) 1 / 4(1 / 2) 1 / 4(1 / 4) 1 / 4(1 / 8) 1 / 4(1 / 16) 1 / 4(1 / 32) 1 / 2(1) 1 / 2(1 / 2) 1 / 2(1 / 4) 1 / 2(1 / 8) 1 / 2(1 / 16) 1 / 2(1 / 32) 1(1) 1(1 / 2) 1(1 / 4) 1(1 / 8) 1(1 / 16) 1(1 / 32)

[0054] (5) According to the chessboard layout in Table 2 above, first dilute the bacterial solution to 106 CFU / mL. Prepare a bacterial solution containing six compound concentration gradients using the diluted bacterial solution from the dosing tank. Inoculate 100 μL per well as indicated in the table. Then, add 4 μL of the methicillin stock solution to the first column and perform a half-fold dilution according to the minimum inhibitory concentration. After dosing, place the 96-well plate in a 37°C incubator for 24 hours.

[0055] (6) Result evaluation: The blank control wells showed no bacterial growth and were clearly clear. The lowest drug concentration that resulted in complete clarity in the wells was the MIC value of the FtsZ protein inhibitor A3 and methicillin (MET) when used in combination. That is, visible clarity with the naked eye indicated 90% inhibition of bacterial growth. The fractional inhibitory concentration (FIC) was calculated using the following formula: FIC = MIC in combination / MIC alone; FIC index (FICI) = FIC A3 +FIC MET .

[0056] Table 3 Synergistic effect of FtsZ protein inhibitor A3 and methicillin (MET) against MRSA

[0057]

[0058]

[0059] The FIC index was less than 0.5, indicating that A3 could improve the sensitivity of methicillin to methicillin-resistant Staphylococcus aureus (MRSA), and the two had a synergistic effect.

[0060] Example 3 Bacterial morphology study

[0061] In this example, an Olympus IX71 inverted fluorescence microscope was used to observe the growth morphology of Bacillus subtilis under the action of the FtsZ protein inhibitor A3.

[0062] B. subtilis 168 was selected as the test bacteria; since the bacterial growth was effectively inhibited when the compound concentration was the MIC value, the experimental concentration was 0.5×MIC to obtain a certain concentration of bacterial suspension for observation.

[0063] The implementation steps are as follows:

[0064] (1) Dilute the cultured bacterial suspension with sterilized MH broth to a concentration of approximately 5×10 5 CFU / mL.

[0065] (2) Add the diluted bacterial suspension and the stock solution of compound A3 to a sterilized 5 mL centrifuge tube to a total volume of 1 mL. The final concentration of the compound is 0.5× the MIC value for inhibition of B. subtilis 168, i.e., 2 μg / mL. The solvent control group is a bacterial suspension without drug treatment.

[0066] (3) Place the centrifuge tube in step (2) in a constant temperature shaker and incubate at 200 rpm and 37°C for 4 to 5 hours until bacterial growth becomes turbid. Observe the bacterial morphology under a 40× lens.

[0067] (4) The experimental results are shown in the attached Figure 1 As shown, compared with the blank group and the solvent control group, the bacterial morphology of the drug-added group was significantly elongated, which preliminarily determined that the compound can act on the target FtsZ protein, causing the bacteria to elongate due to the inability to divide normally, and then die.

[0068] Example 4: FtsZ protein inhibitor A3 interferes with the dynamic polymerization of FtsZ protein

[0069] (1) The in vitro polymerization of FtsZ protein was measured using a multifunctional microplate reader with the wavelength set at 340 nm and the experimental temperature at 25°C.

[0070] (2) Add 50 mM MOPS buffer (pH 6.5) and a final concentration of 6 μM FtsZ protein to a 96-well plate. Add a series of compound concentrations and incubate for 10 min. 1% DMSO is used as a negative control. A control well without FtsZ protein is also set up for background subtraction.

[0071] (3) Add 50 mM KCl, 2 mM MgCl2, and 1 mM CaCl2 and continue scanning for 5 min.

[0072] (4) Add 1 mM GTP solution, continue scanning for 2000 s, and record A 340nm The scanning data were analyzed and processed, and all the scanning data were subjected to corresponding background data subtraction.

[0073] (5) The experimental results are shown in the attached Figure 2 As shown, A3 can promote the dynamic aggregation of FtsZ protein in a concentration-dependent manner.

[0074] It should be noted that the above-described embodiments are to be understood as illustrative and not limiting of the scope of protection of the present invention, which is subject to the claims. It will be apparent to those skilled in the art that non-essential improvements and adjustments to the present invention, without departing from the spirit and scope of the present invention, still fall within the scope of protection of the present invention.

Claims

1. Use of compound A3 in the preparation of drugs against drug-resistant bacteria, characterized in that: The compound A3 is named (1-methyl-2-((Z)-(3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)-4-((E)-4-(4-methylpiperazin-1-yl)phenylvinyl)quinoline-1-iodide, and has a molecular formula of C 32 H 33 IN4S, the structural formula is shown in formula (I): The drug-resistant bacteria are methicillin-resistant Staphylococcus aureus, multidrug-resistant Staphylococcus aureus, vancomycin-intermediate-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, multidrug-resistant Escherichia coli and vancomycin-resistant Enterococcus faecium.

2. Use of compound A3 in the preparation of an anti-Bacillus subtilis drug, characterized in that: The compound A3 is named (1-methyl-2-((Z)-(3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)-4-((E)-4-(4-methylpiperazin-1-yl)phenylvinyl)quinoline-1-iodide, and has a molecular formula of C 32 H 33 IN4S, the structural formula is shown in formula (I):

3. The use according to claim 1 or 2, characterized in that: The medicine further comprises pharmaceutically acceptable excipients or other compatible drugs.

4. The use according to claim 3, characterized in that: The pharmaceutically acceptable excipients are pharmaceutical excipients, including solvents, disintegrants, flavoring agents, preservatives, colorants or adhesives.

5. The use according to claim 1 or 2, characterized in that: The dosage form of the medicine is injection, tablet, pill, capsule, suspension or emulsion.

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