A beta-lactamase inhibitor

Abstract

This application is a divisional application of 202110626296.8. This application belongs to the field of medicine and discloses a β-lactamase inhibitor. The present invention discloses for the first time that thiol-containing compounds (2-methyl-3-tetrahydrofuranthiol) or their derivatives have a good inhibitory effect on β-lactamase (metallo-β-lactamase and / or serine β-lactamase), can protect antibiotics from being degraded by bacteria, increase bacterial sensitivity to antibiotics, and reverse bacterial resistance to antibiotics; at the same time, thiol-containing compounds (2-methyl-3-tetrahydrofuranthiol) and / or their derivatives are used in combination with antibiotics to have a good synergistic effect and can be used as a composite drug to inhibit bacteria.

Description

[0001] This application is a divisional application of the Chinese application with application number 202110626296.8, application date June 4, 2021, and invention name “Application of thiol-containing compounds as and / or in the preparation of β-lactamase inhibitors”. Technical Field

[0002] The invention belongs to the field of medicine, and specifically relates to a beta-lactamase inhibitor. Background Art

[0003] β-lactam antibiotics are the most important and commonly used antibiotics for treating Gram-negative bacterial infections, but the increasing bacterial resistance to them in recent years has caused concerns among humans. Carbapenem, one of the β-lactam antibiotics, is considered to be the last line of defense for humans against bacterial infections. With the emergence of carbapenemases, humans are gradually losing this most important bargaining chip.

[0004] The main reason for bacterial resistance to β-lactam antibiotics is the production of β-lactamases, which can hydrolyze the lactam ring of β-lactam antibiotics and make the antibiotics lose their antibacterial activity. Based on DNA sequence similarity, β-lactamases can be divided into four categories (A, B, C, and D), of which class A, C, and D enzymes are serine-β-lactamases (SBLs), and class B enzymes are metallo-β-lactamases (MBLs), which contain one or two zinc ions in the active site. Metallo-β-lactamases mainly include imipenemases (IMPs), Verona integron-encoded metallo-β-lactamases (VIMs), and New Delhi metallo-β-lactamases (NDMs). Among them, NDM-1-positive bacteria have been widely spread since they were first detected in the early 21st century. Summary of the invention

[0005] The first aspect of the present invention aims to provide the use of thiol-containing compounds or derivatives thereof as and / or in the preparation of β-lactamase inhibitors.

[0006] The second aspect of the present invention aims to provide the use of thiol-containing compounds or their derivatives as and / or in the preparation of drugs for increasing the sensitivity of bacteria to antibiotics.

[0007] The third aspect of the present invention aims to provide the use of antibiotics and sulfhydryl-containing compounds and / or their derivatives in the preparation of drugs for inhibiting bacteria.

[0008] The fourth aspect of the present invention aims to provide a composite medicine comprising an antibiotic and a thiol-containing compound and / or its derivatives.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] The first aspect of the present invention provides the use of a thiol-containing compound or a derivative thereof as and / or in the preparation of a β-lactamase inhibitor.

[0011] Preferably, the thiol-containing compound is at least one of 1,4-butanediol bis(thioglycolate), 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol and 3-thio-1-hexanol; further, at least one of 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol and 1,4-butanediol bis(thioglycolate); further, 1,4-butanediol bis(thioglycolate).

[0012] The molecular formula of 2-pyrazinylethanethiol is C6H8N2S, the CAS number is 35250-53-4, and the structural formula is shown in formula (I).

[0013]

[0014] The molecular formula of 2-methyl-3-tetrahydrofuranthiol is C5H 10 OS, CAS No. is 57124-87-5, and its structural formula is shown in formula (II).

[0015]

[0016] The molecular formula of 3-thio-1-hexanol is C7H 16 OS, CAS No. is 51755-83-0, and its structural formula is shown in formula (III).

[0017]

[0018] The molecular formula of 1,4-butanediol bis(thioglycolate) is C8H 14 O4S2, CAS No. 10193-95-0, and the structural formula is shown in formula (IV).

[0019]

[0020] Preferably, the derivative comprises a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer or prodrug of the thiol-containing compound.

[0021] Preferably, the β-lactamase is at least one of a serine β-lactamase and a metallo β-lactamase; further preferably, the β-lactamase is a metallo β-lactamase.

[0022] Preferably, the metallo-β-lactamase is at least one of IMP-7 type metallo-β-lactamase, NDM-1 type metallo-β-lactamase and VIM-2 type metallo-β-lactamase; further preferably, the metallo-β-lactamase is NDM-1 type metallo-β-lactamase.

[0023] Preferably, the serine β-lactamase is KPC-2 type serine β-lactamase.

[0024] Preferably, the source of the β-lactamase includes extraction from nature or preparation from genetically engineered strains.

[0025] The second aspect of the present invention provides the use of thiol-containing compounds or derivatives thereof as and / or in the preparation of drugs for increasing the sensitivity of bacteria to antibiotics.

[0026] Preferably, the thiol-containing compound is at least one of 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol and 1,4-butanediol bis(thioglycolate); further, at least one of 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol and 3-thio-1-hexanol; further, at least one of 2-pyrazinylethanethiol and 2-methyl-3-tetrahydrofuranthiol.

[0027] The molecular formula of 2-pyrazinylethanethiol is C6H8N2S, the CAS number is 35250-53-4, and the structural formula is shown in formula (I).

[0028] The molecular formula of 2-methyl-3-tetrahydrofuranthiol is C5H 10 OS, CAS No. is 57124-87-5, and its structural formula is shown in formula (II).

[0029] The molecular formula of 3-thio-1-hexanol is C7H 16 OS, CAS No. is 51755-83-0, and its structural formula is shown in formula (III).

[0030] The molecular formula of 1,4-butanediol bis(thioglycolate) is C8H 14 O4S2, CAS No. 10193-95-0, and the structural formula is shown in formula (IV).

[0031] Preferably, the derivative comprises a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer or prodrug of the thiol-containing compound.

[0032] Preferably, the bacterium is a drug-resistant bacterium expressing metallo-β-lactamase and / or serine-β-lactamase; further preferably, the bacterium is at least one of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa.

[0033] Preferably, the antibiotic is a β-lactam antibiotic; further preferably, the antibiotic is at least one of penicillin antibiotics, cephalosporin antibiotics, cephamycin antibiotics, thiomycin antibiotics and carbapenem antibiotics; even more preferably, the antibiotic is at least one of meropenem, imipenem, ertapenem, cephalexin, cefuroxime, cefdinir, ceftriaxone, ceftazidime, ampicillin and amoxicillin.

[0034] Preferably, the metallo-β-lactamase is at least one of IMP-7 type metallo-β-lactamase, NDM-1 type metallo-β-lactamase and VIM-2 type metallo-β-lactamase; further preferably, the metallo-β-lactamase is at least one of IMP-7 type metallo-β-lactamase and NDM-1 type metallo-β-lactamase; further preferably, the metallo-β-lactamase is NDM-1 type metallo-β-lactamase.

[0035] Preferably, the serine β-lactamase is KPC-2 type serine β-lactamase.

[0036] The third aspect of the present invention provides the use of antibiotics and thiol-containing compounds and / or their derivatives in the preparation of drugs for inhibiting bacteria.

[0037] Preferably, the thiol-containing compound is at least one of 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol and 1,4-butanediol bis(thioglycolate); further, at least one of 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol and 3-thio-1-hexanol; further, at least one of 2-methyl-3-tetrahydrofuranthiol and 3-thio-1-hexanol.

[0038] The molecular formula of 2-pyrazinylethanethiol is C6H8N2S, the CAS number is 35250-53-4, and the structural formula is shown in formula (I).

[0039] The molecular formula of 2-methyl-3-tetrahydrofuranthiol is C5H 10 OS, CAS No. is 57124-87-5, and its structural formula is shown in formula (II).

[0040] The molecular formula of 3-thio-1-hexanol is C7H 16 OS, CAS No. is 51755-83-0, and its structural formula is shown in formula (III).

[0041] The molecular formula of 1,4-butanediol bis(thioglycolate) is C8H 14 O4S2, CAS No. 10193-95-0, and the structural formula is shown in formula (IV).

[0042] Preferably, the derivative comprises a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer or prodrug of the thiol-containing compound.

[0043] Preferably, the antibiotic is a β-lactam antibiotic; further preferably, the antibiotic is at least one of penicillin antibiotics, cephalosporin antibiotics, cephamycin antibiotics, thiomycin antibiotics and carbapenem antibiotics; even more preferably, the antibiotic is at least one of meropenem, imipenem, ertapenem, cephalexin, cefuroxime, cefdinir, ceftriaxone, ceftazidime, ampicillin and amoxicillin.

[0044] Preferably, the bacterium is a drug-resistant bacterium expressing metallo-β-lactamase and / or serine-β-lactamase; further preferably, the bacterium is at least one of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa.

[0045] Preferably, the metallo-β-lactamase is at least one of IMP-7 type metallo-β-lactamase, NDM-1 type metallo-β-lactamase and VIM-2 type metallo-β-lactamase; further preferably, the metallo-β-lactamase is at least one of IMP-7 type metallo-β-lactamase and NDM-1 type metallo-β-lactamase; further preferably, the metallo-β-lactamase is NDM-1 type metallo-β-lactamase.

[0046] Preferably, the serine β-lactamase is KPC-2 type serine β-lactamase.

[0047] A fourth aspect of the present invention provides a medicine comprising:

[0048] (1) antibiotics; and

[0049] (2) Thiol-containing compounds and / or their derivatives.

[0050] Preferably, the thiol-containing compound is at least one of 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol and 1,4-butanediol bis(thioglycolate); further, at least one of 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol and 3-thio-1-hexanol; further, at least one of 2-pyrazinylethanethiol and 2-methyl-3-tetrahydrofuranthiol.

[0051] The molecular formula of 2-pyrazinylethanethiol is C6H8N2S, the CAS number is 35250-53-4, and the structural formula is shown in formula (I).

[0052] The molecular formula of 2-methyl-3-tetrahydrofuranthiol is C5H 10 OS, CAS No. is 57124-87-5, and its structural formula is shown in formula (II).

[0053] The molecular formula of 3-thio-1-hexanol is C7H 16 OS, CAS No. is 51755-83-0, and its structural formula is shown in formula (III).

[0054] The molecular formula of 1,4-butanediol bis(thioglycolate) is C8H 14 O4S2, CAS No. 10193-95-0, and the structural formula is shown in formula (IV).

[0055] Preferably, the derivative comprises a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer or prodrug of the thiol-containing compound.

[0056] Preferably, the antibiotic is a β-lactam antibiotic; further preferably, the antibiotic is at least one of penicillin antibiotics, cephalosporin antibiotics, cephamycin antibiotics, thiomycin antibiotics and carbapenem antibiotics; even more preferably, the antibiotic is at least one of meropenem, imipenem, ertapenem, cephalexin, cefuroxime, cefdinir, ceftriaxone, ceftazidime, ampicillin and amoxicillin.

[0057] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0058] Preferably, the auxiliary materials include at least one of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, and a lubricant.

[0059] Preferably, the drug preparation types include solid preparations, liquid preparations and semisolid preparations.

[0060] Preferably, the solid preparation includes tablets, granules, powders and capsules.

[0061] Preferably, the liquid preparation comprises an injection.

[0062] Preferably, the semisolid preparation includes ointments and creams.

[0063] The beneficial effects of the present invention are:

[0064] The invention discloses for the first time the use of thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol and 1,4-butanediol bis(thioglycolate)) or their derivatives as and / or in the preparation of metal β-lactamase inhibitors. The thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol and 1,4-butanediol bis(thioglycolate)) or their derivatives have good inhibitory effects on β-lactamase (metal β-lactamase and / or serine β-lactamase), can protect antibiotics from being degraded by bacteria, improve bacterial sensitivity to antibiotics, and reverse bacterial resistance to antibiotics; meanwhile, the thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol) and / or their derivatives are used in combination with antibiotics to have good synergistic effects and can be used as composite drugs for inhibiting bacteria. DETAILED DESCRIPTION

[0065] The present invention is further described in detail below through specific examples.

[0066] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0067] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The materials and reagents used in the examples are commercially available unless otherwise specified.

[0068] Example 1 Determination of the inhibitory activity of sulfhydryl compounds on β-lactamase

[0069] After the substrate is hydrolyzed by the enzyme, the absorbance value will decrease. Therefore, the change in absorbance can be used to characterize the degree of substrate hydrolysis, thereby judging the activity of the enzyme. Meropenem (50 μM) was used as the reporting substrate, and the absorbance change of the substrate after being hydrolyzed by metallo-β-lactamase was measured at a wavelength of 300 nm. Metallo-β-lactamases include NDM-1, VIM-2 and IMP-7, with final concentrations of 2nM, 4nM and 5nM, respectively. The buffer is 50mM HEPES (4-hydroxyethylpiperazineethanesulfonic acid), and ZnSO4 (final concentration of 0.1mM), Triton X-100 (final concentration of 0.01% (v / v)), and bovine serum albumin (BSA, final concentration of 0.1μg / mL) were added. The pH was 7.2, and the reaction temperature was 25°C. The specific experimental method is as follows:

[0070] 1. Determination of the inhibitory activity of thiol-containing compounds against IMP-7 metallo-β-lactamase

[0071] (1) 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol and 1,4-butanediol bis(thioglycolate) (captopril was added to the control group, purchased from Shanghai Yuanye Biotechnology Co., Ltd., batch number S30916) were dissolved in HEPES buffer and prepared into different concentrations (0.1, 0.5, 1, 5, 10, 50, 100, 300, 500 μM, respectively). Three replicate wells were set for each concentration, and 10 μL of IMP-7 metallo-β-lactamase solution (final concentration of 5 nM) was added. The cells were incubated at 25°C for 15 min to allow 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 2, 3-thio-1-hexanol and 1,4-butanediol bis(thioglycolate) and captopril to fully bind to the enzyme.

[0072] (2) The system was transferred to a quartz cuvette, 50 μL of meropenem (final concentration 50 μM) was added, and the change in absorbance was quickly measured and the data was recorded.

[0073] (3) Calculate the inhibition rate of different concentrations of 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol, 1,4-butanediol bis(thioglycolate) and captopril on IMP-7 metallo-β-lactamase, plot the concentration of the compound against the residual activity of IMP-7 metallo-β-lactamase, and calculate the IC by fitting the curve. 50 value, and Ki=IC 50 The Ki value was calculated by using the equation: / (1+[S] / Km) (where [S] is the substrate concentration and Km is the Michaelis constant of the enzyme).

[0074] 2. Determination of the inhibitory activity of thiol-containing compounds against NDM-1 metallo-β-lactamase

[0075] (1) 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol and 1,4-butanediol bis(thioglycolate) (captopril was added to the control group, purchased from Shanghai Yuanye Biotechnology Co., Ltd., batch number S30916) were dissolved in HEPES buffer and prepared into different concentrations (0.1, 0.5, 1, 5, 10, 50, 100, 300, 500 μM, respectively). Three replicate wells were set for each concentration, and 10 μL of NDM-1 metallo-β-lactamase solution (final concentration was 2 nM) was added. The cells were incubated at 25°C for 15 min to allow 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 2, 3-thio-1-hexanol, 1,4-butanediol bis(thioglycolate) and captopril to fully bind to the enzyme.

[0076] (2) The system was transferred to a quartz cuvette, 50 μL of meropenem (final concentration 50 μM) was added, and the change in absorbance was quickly measured and the data was recorded.

[0077] (3) Calculate the inhibition rate of different concentrations of 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol, 1,4-butanediol bis(thioglycolate) and captopril on NDM-1 metallo-β-lactamase, plot the concentration of the compound against the residual activity of NDM-1 metallo-β-lactamase, and calculate the IC by fitting the curve. 50 value and calculate the Ki value.

[0078] 3. Determination of the inhibitory activity of thiol-containing compounds against VIM-2 metallo-β-lactamase

[0079] (1) 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol and 1,4-butanediol bis(thioglycolate) (captopril was added to the control group, purchased from Shanghai Yuanye Biotechnology Co., Ltd., batch number S30916) were dissolved in HEPES buffer and prepared into different concentrations (0.1, 0.5, 1, 5, 10, 50, 100, 300, 500 μM, respectively). Three replicate wells were set for each concentration, and 10 μL of VIM-2 metallo-β-lactamase solution (final concentration of 4 nM) was added. The cells were incubated at 25°C for 15 min to allow 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol and 1,4-butanediol bis(thioglycolate) (captopril) to fully bind to the enzyme.

[0080] (2) The system was transferred to a quartz cuvette, 50 μL of meropenem (final concentration 50 μM) was added, and the change in absorbance was quickly measured and the data was recorded.

[0081] (3) Calculate the inhibition rate of different concentrations of 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol, 1,4-butanediol bis(thioglycolate) and captopril on VIM-2 metallo-β-lactamase, plot the concentration of the compound against the residual activity of VIM-2 metallo-β-lactamase, and calculate the IC by fitting the curve. 50 value and calculate the Ki value.

[0082] The results of the inhibitory activities of thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol and 1,4-butanediol bis(thioglycolate)) and captopril against IMP-7, NDM-1 and VIM-2 metallo-β-lactamases are shown in Table 1: The inhibitory activities of 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol and 1,4-butanediol bis(thioglycolate) against NDM-1 metallo-β-lactamases The inhibitory effect of 1,4-butanediol bis(thioglycolate) on NDM-1 metallo-β-lactamase was better than that of captopril.

[0083] Table 1 IC values ​​of sulfhydryl compounds and captopril against IMP-7, NDM-1 and VIM-2 metallo-β-lactamases 50 (μM) and Ki (μM) Example 2 Evaluation of the effect of thiol-containing compounds combined with meropenem in inhibiting β-lactamase-producing drug-resistant bacteria

[0084] The minimum inhibitory concentration (MIC) of thiol-containing compounds combined with meropenem against β-lactamase-resistant strains was determined by the microbroth dilution method. The E. coli BL21 (DE3) / pMAL-c5x-IMP-7 used in the experiment was purchased from Shanghai Shenggong Biotechnology Co., Ltd., E. coli BL21 (DE3) / pET24a-VIM-2 and E. coli BL21 (DE3) / pET26b-NDM-1 were donated by Professor Yang Kewu of Northwest University and have been published in the literature: Zhang Yuejuan, Characterization and Inhibition of Antibiotic Resistance Target Protein Metallo-β-lactamase and Its Resistant Bacteria [D], Northwest University, 2019; it also includes the clinical isolate E. coli BAA-2452 (bla purchased from Beijing Biobo Biotechnology Co., Ltd. NDM-1 ) and E.coli BAA-2340(bla KPC-2).

[0085] FICI is used to determine the interaction between two drugs when used in combination and is defined according to the following equation: FICI = FIC A +FIC B =C A / MIC A +C B / MIC B , where MIC A and MIC B are the MIC values ​​of compounds A and B used alone, while C A and C B It is the drug concentration of compound A and B in the effective combination. If FICI≤0.5, the two drugs are considered to have a synergistic effect, 0.5<FICI≤4 indicates that the synergistic effect of the two drugs is weak or has no relevant effect, and FICI≥4 indicates that the two drugs have an antagonistic effect. The smaller the FICI, the stronger the synergistic effect of the drugs.

[0086] The specific experimental methods are as follows:

[0087] 1. Evaluation of the efficacy of thiol-containing compounds combined with meropenem in inhibiting IMP-7-producing drug-resistant bacteria

[0088] (1) Under aseptic operation conditions, the ultra-low temperature preserved strain (E. coli BL21 (DE3) / pMAL-c5x-IMP-7 bacteria) was inoculated into a sterile LB solid culture medium and cultured in a 37°C constant temperature incubator overnight. A single colony was picked and transferred to 3 mL LB liquid culture medium (containing 50 mg / mL ampicillin), and cultured in a 37°C constant temperature incubator until the logarithmic growth phase to obtain a bacterial suspension; the concentration of the bacterial suspension was adjusted to 0.5 McFarland concentration using a McFarland turbidimeter, and the LB liquid culture medium was diluted 100 times. The number of bacteria was about 1×10 6 CFU / mL.

[0089] (2) Add 100 μL into columns 2 to 12 of a 96-well plate. LB liquid culture medium, and add 100 μL of meropenem solution (256 μg / mL) or inhibitor (sulfhydryl compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis(thioglycolate), 256 μg / mL) in the first column, and after the second column of drug solution is fully mixed, 100 μL is drawn and added to the third column and mixed again, and the drug solution is diluted in turn by this doubling dilution method to obtain a drug concentration of 0.0625-128 μg / mL; 100 μL of the diluted bacterial solution is added to each well to determine the MIC of meropenem or sulfhydryl compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis(thioglycolate)) against IMP-7-producing drug-resistant bacteria alone, and three replicate wells are set for each concentration.

[0090] (3) The drug solution was diluted in both horizontal and vertical directions on a 96-well plate. The horizontal row was a gradient dilution of meropenem. The method was the same as step (2), but the volume of LB liquid culture medium and meropenem added was 50 μL (the final concentration of meropenem was 0.0625-128 μg / mL), and 50 μL of inhibitors of different concentrations (sulfhydryl compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis(thioglycolate)), the final concentration was 2-128 μg / mL; 100 μL of the diluted bacterial solution was added to each well to determine the MIC of meropenem combined with inhibitors (thiol-containing compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis(thioglycolate)) against IMP-7-producing resistant bacteria, and three replicate wells were set for each concentration.

[0091] (4) Each experiment was set up with three parallel control groups: Escherichia coli ATCC25922 was used as the quality control standard, captopril was used as the positive control, and sterile wells and drug-free wells were set up. The 96-well plate was placed in a 37°C constant temperature incubator for 24 h, and the results were observed and the MIC values ​​were recorded.

[0092] 2. Evaluation of the efficacy of thiol-containing compounds combined with meropenem in inhibiting VIM-2-producing resistant bacteria

[0093] (1) Under aseptic operation conditions, the ultra-low temperature preserved strain (E. coli BL21 (DE3) / pET24a-VIM-2 bacteria) was inoculated into sterile LB solid culture medium and cultured in a 37°C constant temperature incubator overnight. A single colony was picked and transferred to 3 mL LB liquid culture medium (containing 50 mg / mL kanamycin) and cultured in a 37°C constant temperature incubator until the logarithmic growth phase to obtain a bacterial suspension; the concentration of the bacterial suspension was adjusted to 0.5 McFarland concentration using a McFarland turbidimeter, and the LB liquid culture medium was diluted 100 times. The number of bacteria was about 1×10 6 CFU / mL.

[0094] (2) Add 100 μL into columns 2 to 12 of a 96-well plate. LB liquid culture medium, and add 100 μL of meropenem solution (256 μg / mL) or inhibitor (sulfhydryl compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis(thioglycolate), 256 μg / mL) in the first column, and after the second column of drug solution is fully mixed, 100 μL is drawn and added to the third column and mixed again, and the drug solution is diluted in turn by this doubling dilution method to obtain a drug concentration of 0.0625-128 μg / mL; 100 μL of the diluted bacterial solution is added to each well to determine the MIC of meropenem or sulfhydryl compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis(thioglycolate)) against VIM-2-resistant bacteria, and three replicate wells are set for each concentration.

[0095] (3) The drug solution was diluted in both horizontal and vertical directions on a 96-well plate. The horizontal row was a gradient dilution of meropenem. The method was the same as step (2), but the volume of LB liquid culture medium and meropenem added was 50 μL (the final concentration of meropenem was 0.0625-128 μg / mL), and 50 μL of inhibitors of different concentrations (sulfhydryl compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis(thioglycolate)), the final concentration was 2-128 μg / mL; 100 μL of diluted bacterial solution was added to each well to determine the MIC of meropenem combined with inhibitors (thiol-containing compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis(thioglycolate)) against VIM-2-producing resistant bacteria, and three replicate wells were set for each concentration.

[0096] (4) Each experiment was set up with three parallel control groups: Escherichia coli ATCC25922 was used as the quality control standard, captopril was used as the positive control, and sterile wells and drug-free wells were set up. The 96-well plate was placed in a 37°C constant temperature incubator for 24 h, and the results were observed and the MIC values ​​were recorded.

[0097] 3. Evaluation of the efficacy of thiol-containing compounds combined with meropenem in inhibiting NDM-1-producing resistant bacteria

[0098] (1) Under aseptic operation conditions, the ultra-low temperature preserved strain (E. coli BL21 (DE3) / pET26b-NDM-1 bacteria) was inoculated into sterile LB solid culture medium and cultured in a 37°C constant temperature incubator overnight. A single colony was picked and transferred to 3 mL LB liquid culture medium (containing 50 mg / mL kanamycin) and cultured in a 37°C constant temperature incubator until the logarithmic growth phase to obtain a bacterial suspension; the concentration of the bacterial suspension was adjusted to 0.5 McFarland concentration using a McFarland turbidimeter, and the LB liquid culture medium was diluted 100 times. The number of bacteria was about 1×10 6 CFU / mL.

[0099] (2) Add 100 μL into columns 2 to 12 of a 96-well plate. LB liquid culture medium, and add 100 μL of meropenem solution (256 μg / mL) or inhibitor (sulfhydryl compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis(thioglycolate), 256 μg / mL) in the first column, and after the second column of drug solution is fully mixed, 100 μL is drawn and added to the third column and mixed again, and the drug solution is diluted in turn by this doubling dilution method to obtain a drug concentration of 0.0625-128 μg / mL; 100 μL of the diluted bacterial solution is added to each well to determine the MIC of meropenem or sulfhydryl compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis(thioglycolate)) against NDM-1-resistant bacteria, and three replicate wells are set for each concentration.

[0100] (3) The drug solution was diluted in both horizontal and vertical directions on a 96-well plate. The horizontal row was a gradient dilution of meropenem. The method was the same as step (2), but the volume of LB liquid culture medium and meropenem added was 50 μL (the final concentration of meropenem was 0.0625-128 μg / mL), and 50 μL of inhibitors of different concentrations (sulfhydryl compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis(thioglycolate)), the final concentration was 2-128 μg / mL; 100 μL of diluted bacterial solution was added to each well to determine the MIC of meropenem combined with inhibitors (thiol-containing compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis(thioglycolate)) against NDM-1-producing resistant bacteria, and three replicate wells were set for each concentration.

[0101] (4) Each experiment was set up with three parallel control groups: Escherichia coli ATCC25922 was used as the quality control standard, captopril was used as the positive control, and sterile wells and drug-free wells were set up. The 96-well plate was placed in a 37°C constant temperature incubator for 24 h, and the results were observed and the MIC values ​​were recorded.

[0102] 4. Combination of thiol-containing compounds and meropenem inhibits the clinical isolate E. coli BAA-2452 (bla NDM-1 )Evaluation of the effect

[0103] (1) Under aseptic conditions, the ultra-low temperature stored strain (clinical isolate E. coli BAA-2452 (bla NDM-1 )) was inoculated into sterile LB solid medium and cultured overnight in a 37°C constant temperature incubator. A single colony was picked and transferred to 3 mL LB liquid medium and cultured in a 37°C constant temperature incubator until the logarithmic growth phase to obtain a bacterial suspension. The concentration of the bacterial suspension was adjusted to 0.5 McFarland concentration using a McFarland turbidimeter. The LB liquid medium was diluted 100 times and the number of bacteria was about 1×10 6 CFU / mL.

[0104] (2) Add 100 μL into columns 2 to 12 of a 96-well plate. LB liquid culture medium, and add 100 μL of meropenem solution (256 μg / mL) or inhibitor (sulfhydryl compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis(thioglycolate), 256 μg / mL) in the first column, and after the second column of the drug solution is fully mixed, 100 μL is drawn and added to the third column and mixed again. The drug solution is diluted in turn by this doubling dilution method to obtain a drug concentration of 0.0625-128 μg / mL; 100 μL of the diluted bacterial solution is added to each well to determine the effect of using meropenem alone or sulfhydryl compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis(thioglycolate)) on the clinical isolate E. coli BAA-2452 (bla NDM-1 ) and three replicate wells were set for each concentration.

[0105] (3) The drug solution was diluted in both horizontal and vertical directions on a 96-well plate. The horizontal rows were gradient dilutions of meropenem. The method was the same as step (2), but the volume of LB liquid culture medium and meropenem added was 50 μL (the final concentration of meropenem was 0.0625-128 μg / mL), and 50 μL of inhibitors (containing thiol compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis(thioglycolate)) with different concentrations diluted in advance were added in the vertical columns, with a final concentration of 2-128 μg / mL; 100 μL of the diluted bacterial solution was added to each well to determine the effect of meropenem combined with inhibitors (containing thiol compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis(thioglycolate)) on clinical isolates of E. coli. BAA-2452(bla NDM-1 ) and three replicate wells were set for each concentration.

[0106] (4) Each experiment was set up with three parallel control groups: Escherichia coli ATCC25922 was used as the quality control standard, captopril was used as the positive control, and sterile wells and drug-free wells were set up. The 96-well plate was placed in a 37°C constant temperature incubator for 24 h, and the results were observed and the MIC values ​​were recorded.

[0107] 5. Combination of thiol-containing compounds and meropenem inhibits the clinical isolate E. coli BAA-2340 (bla KPC-2 )Evaluation of the effect

[0108] (1) Under aseptic conditions, the ultra-low temperature stored strain (clinical isolate E. coli BAA-2340 (bla KPC-2 )) was inoculated into sterile LB solid medium and cultured overnight in a 37°C constant temperature incubator. A single colony was picked and transferred to 3 mL LB liquid medium and cultured in a 37°C constant temperature incubator until the logarithmic growth phase to obtain a bacterial suspension. The concentration of the bacterial suspension was adjusted to 0.5 McFarland concentration using a McFarland turbidimeter. The LB liquid medium was diluted 100 times and the number of bacteria was about 1×10 6 CFU / mL.

[0109] (2) Add 100 μL into columns 2 to 12 of a 96-well plate. LB liquid culture medium, and add 100 μL of meropenem solution (256 μg / mL) or inhibitor (sulfhydryl compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis(thioglycolate), 256 μg / mL) in the first column, and after the second column of the drug solution is fully mixed, 100 μL is drawn and added to the third column and mixed again. The drug solution is diluted in turn by this doubling dilution method to obtain a drug concentration of 0.0625-128 μg / mL; 100 μL of the diluted bacterial solution is added to each well to determine the effect of using meropenem alone or sulfhydryl compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis(thioglycolate)) on the clinical isolate E. coli BAA-2340 (bla KPC-2 ) and three replicate wells were set for each concentration.

[0110] (3) The drug solution was diluted in both horizontal and vertical directions on a 96-well plate. The horizontal rows were gradient dilutions of meropenem. The method was the same as step (2), but the volume of LB liquid culture medium and meropenem added was 50 μL (the final concentration of meropenem was 0.0625-128 μg / mL), and 50 μL of inhibitors (containing thiol compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis(thioglycolate)) with different concentrations diluted in advance were added in the vertical columns, with a final concentration of 2-128 μg / mL; 100 μL of the diluted bacterial solution was added to each well to determine the effect of meropenem combined with inhibitors (containing thiol compounds: 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis(thioglycolate)) on clinical isolates of E. coli. BAA-2340(bla KPC-2 ) and three replicate wells were set for each concentration.

[0111] (4) Each experiment was set up with three parallel control groups: Escherichia coli ATCC25922 was used as the quality control standard, captopril was used as the positive control, and sterile wells and drug-free wells were set up. The 96-well plate was placed in a 37°C constant temperature incubator for 24 h, and the results were observed and the MIC values ​​were recorded.

[0112] The results of the antibacterial activity of meropenem combined with thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol and 1,4-butanediol bis(thioglycolate)) against resistant bacteria expressing NDM-1, VIM-2, IMP-7 or KPC-2 are shown in Table 2: Inhibitors (thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol, 1,4-butanediol bis(thioglycolate)), captopril) can improve the antibacterial activity of meropenem: when the inhibitor concentration is 128 μg / mL, combined use with meropenem can improve the antibacterial effect of meropenem against a variety of resistant bacteria expressing β-lactamases, and compared with the use of meropenem alone, the combined use can effectively reduce the MIC value of meropenem against resistant strains, which can be reduced by up to 8 times.

[0113] The FICI values ​​of thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol and 1,4-butanediol bis(thioglycolate)) or captopril combined with meropenem against resistant bacteria expressing β-lactamase are shown in Table 3: The FICI values ​​of thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol) combined with meropenem against resistant bacteria expressing NDM-1 type metallo-β-lactamase were all less than or equal to 0.5, indicating that the two had good synergistic effects; 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol, 1,4-butanediol bis(thioglycolate) The FICI value of the combination of 2-pyrazinylethanethiol and meropenem for resistant bacteria expressing IMP-7 metallo-β-lactamase was less than or equal to 0.5, indicating that the two had a good synergistic effect; the FICI value of the combination of 2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol and meropenem for resistant bacteria expressing KPC-2 serine β-lactamase was less than or equal to 0.5, indicating that the two had a good synergistic effect, while the FICI value of the combination of captopril and meropenem for resistant bacteria expressing KPC-2 serine β-lactamase was greater than 1, indicating that the two had no synergistic effect.

[0114] The above results show that: in combination with meropenem, thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol) have effective synergistic antibacterial activity against resistant bacteria expressing NDM-1 type metallo-β-lactamase, 2-pyrazinylethanethiol has effective synergistic antibacterial activity against resistant bacteria expressing IMP-7 type metallo-β-lactamase, and thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol) have effective synergistic antibacterial activity against resistant bacteria expressing KPC-2 type serine β-lactamase, indicating that thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol) have effective synergistic antibacterial activity against resistant bacteria expressing KPC-2 type serine β-lactamase. The thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol) can be used as NDM-1 type metallo-β-lactamase inhibitors, 2-pyrazinylethanethiol can be used as IMP-7 type metallo-β-lactamase inhibitors, and the thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol) can be used as KPC-2 type serine β-lactamase inhibitors, which can reverse the drug resistance of carbapenem-resistant bacteria, effectively protect meropenem from being hydrolyzed by NDM-1 type, IMP-7 type, and KPC-2 type metallo-β-lactamases, and improve the antibacterial activity of meropenem against resistant bacteria producing NDM-1 type, IMP-7 type, and KPC-2 type metallo-β-lactamases. Therefore, the thiol-containing compounds (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol) can be used as β-lactamase inhibitors and prepared into a composite preparation with β-lactam antibiotics.

[0115] Table 2 MIC (μg / mL) of thiol-containing compounds or captopril combined with meropenem against resistant bacteria expressing β-lactamase

[0116]

[0117]

[0118] Table 3 Synergistic antimicrobial index (FICI) of thiol-containing compounds or captopril combined with meropenem against resistant bacteria expressing β-lactamase

[0119]

[0120] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of a thiol-containing compound or a pharmaceutically acceptable salt thereof in the preparation of a β-lactamase inhibitor, wherein the thiol-containing compound is 2-methyl-3-tetrahydrofuranthiol; The β-lactamase is at least one of serine β-lactamase and metallo β-lactamase; The metallo-β-lactamase is at least one of IMP-7 metallo-β-lactamase, NDM-1 metallo-β-lactamase and VIM-2 metallo-β-lactamase; The serine β-lactamase is KPC-2 type serine β-lactamase.

2. Use of a thiol-containing compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for increasing the sensitivity of bacteria to antibiotics, wherein the thiol-containing compound is 2-methyl-3-tetrahydrofuranthiol; The antibiotic is a β-lactam antibiotic; The bacteria are drug-resistant bacteria expressing metallo-β-lactamase and / or serine β-lactamase; The metallo-β-lactamase is at least one of IMP-7 metallo-β-lactamase, NDM-1 metallo-β-lactamase and VIM-2 metallo-β-lactamase; The serine β-lactamase is KPC-2 type serine β-lactamase.

3. Use of an antibiotic and a thiol-containing compound and / or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting bacteria, wherein the thiol-containing compound is 2-methyl-3-tetrahydrofuranthiol; The antibiotic is a β-lactam antibiotic; The bacteria are drug-resistant bacteria expressing metallo-β-lactamase and / or serine β-lactamase; The metallo-β-lactamase is at least one of IMP-7 metallo-β-lactamase, NDM-1 metallo-β-lactamase and VIM-2 metallo-β-lactamase; The serine β-lactamase is KPC-2 type serine β-lactamase.

4. A drug for inhibiting bacteria, comprising: (1) antibiotics; and (2) sulfhydryl-containing compounds and / or pharmaceutically acceptable salts thereof; The thiol-containing compound is 2-methyl-3-tetrahydrofuranthiol; The antibiotic is a β-lactam antibiotic; The bacteria are drug-resistant bacteria expressing metallo-β-lactamase and / or serine β-lactamase; The metallo-β-lactamase is at least one of IMP-7 metallo-β-lactamase, NDM-1 metallo-β-lactamase and VIM-2 metallo-β-lactamase; The serine β-lactamase is KPC-2 type serine β-lactamase.

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