CN113425726B - Application of sulfhydryl-containing compound as and/or in preparation of beta-lactamase inhibitor - Google Patents

Application of sulfhydryl-containing compound as and/or in preparation of beta-lactamase inhibitor Download PDF

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CN113425726B
CN113425726B CN202110626296.8A CN202110626296A CN113425726B CN 113425726 B CN113425726 B CN 113425726B CN 202110626296 A CN202110626296 A CN 202110626296A CN 113425726 B CN113425726 B CN 113425726B
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lactamase
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pyrazinylethylthiol
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CN113425726A (en
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石磊
何羡霞
张冰
姜志辉
杨琰
袁进
蔡敏捷
蔡学究
李小荣
廖扬
许文平
黄雪峰
陈龙浩
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Southern Theater Command General Hospital of PLA
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Abstract

The invention belongs to the field of medicines, and discloses application of a sulfhydryl-containing compound in serving as and/or preparing a beta-lactamase inhibitor. The invention discloses that the sulfhydryl-containing compound (2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuryl mercaptan, 3-thio-1-hexanol and 1,4-butanediol bis (mercaptoacetate)) or the derivative thereof has good inhibition effect on beta-lactamase (metallo beta-lactamase and/or serine beta-lactamase), can protect antibiotics from being degraded by bacteria, improve the sensitivity of the bacteria to the antibiotics and reverse the drug resistance of the bacteria to the antibiotics for the first time; meanwhile, the sulfhydryl-containing compound (2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuryl mercaptan, 3-thio-1-hexanol) and/or the derivative thereof has good synergistic effect when being combined with antibiotics, and can be used as a compound medicament for inhibiting bacteria.

Description

Application of sulfhydryl-containing compound as and/or in preparation of beta-lactamase inhibitor
Technical Field
The invention belongs to the field of medicines, and particularly relates to application of a sulfhydryl-containing compound in serving as and/or preparing a beta-lactamase inhibitor.
Background
Beta-lactam antibiotics are the most important and commonly used antibiotics for the treatment of gram-negative bacterial infections, but the increasing resistance of bacteria to them has raised concerns in humans in recent years. Carbapenems, one of the β -lactam antibiotics, are considered to be the last line of defense against bacterial infections in humans, and with the advent of carbapenemases, humans gradually lost this most important counter.
The most significant cause of bacterial resistance to beta-lactam antibiotics is the production of beta-lactamase enzymes, which can inactivate the antibiotic activity by hydrolyzing the lactam ring of the beta-lactam antibiotic. Beta-lactamases can be classified into four classes (classes A, B, C, D) based on DNA sequence similarity, wherein the enzymes of classes A, C and D are serine-beta-lactamases (SBLs), the enzymes of class B are metallo-beta-lactamases (MBLs), and the active site contains one or two zinc ions. Metallo-beta-lactamases mainly include Imipenemase (IMPs), verona integrant-encoded metallo-beta-lactamases (VIMs) and New Delhi metallo-beta-lactamases (NDMs). Among them, NDM-1 positive bacteria have been widely spread since the first detection in the early century.
Disclosure of Invention
The object of the first aspect of the present invention is to provide a thiol-group-containing compound or a derivative thereof for use as and/or for preparing a beta-lactamase inhibitor.
The object of the second aspect of the present invention is to provide the use of a thiol-group-containing compound or a derivative thereof as and/or for the manufacture of a medicament for increasing the sensitivity of bacteria to antibiotics.
The third aspect of the present invention is to provide the use of antibiotics and sulfhydryl-containing compounds and/or derivatives thereof in the preparation of medicaments for inhibiting bacteria.
The fourth aspect of the present invention is to provide a combination drug comprising an antibiotic and a thiol-group-containing compound and/or a derivative thereof.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided the use of a thiol-group-containing compound or a derivative thereof as and/or in the preparation of an inhibitor of beta-lactamase.
Preferably, the mercapto-containing compound is at least one of 1,4-butanediol bis (mercaptoacetate), 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofurthiol and 3-thio-1-hexanol; further at least one of 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol and 1,4-butanediol bis (mercaptoacetate); further 1,4-butanediol bis (mercaptoacetate).
2-pyrazinylethylthiol having the formula C 6 H 8 N 2 S, the CAS number is 35250-53-4, and the structural formula is shown as the formula (I).
Figure BDA0003101274780000021
The molecular formula of the 2-methyl-3-tetrahydrofuran thiol is C 5 H 10 OS, CAS number 57124-87-5, the structural formula is shown as formula (II).
Figure BDA0003101274780000022
Molecular formula of 3-thio-1-hexanolIs C 7 H 16 OS, CAS number 51755-83-0, the constitutional formula is shown as formula (III).
Figure BDA0003101274780000023
1,4-butanediol bis (mercaptoacetate) has the molecular formula C 8 H 14 O 4 S 2 The CAS number is 10193-95-0, and the structural formula is shown as the formula (IV).
Figure BDA0003101274780000031
Preferably, the derivative includes a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer or prodrug of the thiol-containing compound.
Preferably, the beta-lactamase is at least one of a serine beta-lactamase and a metallo beta-lactamase; further preferably, the beta-lactamase is a metallo beta-lactamase.
Preferably, the metallo-beta-lactamase is at least one of an IMP-7 type metallo-beta-lactamase, an NDM-1 type metallo-beta-lactamase and a VIM-2 type metallo-beta-lactamase; further preferably, the metallo-beta-lactamase is an NDM-type 1 metallo-beta-lactamase.
Preferably, the serine beta-lactamase is a KPC-2 type serine beta-lactamase.
Preferably, the source of the beta-lactamase comprises extraction in nature or preparation from a genetically engineered strain.
In a second aspect of the invention, there is provided the use of a thiol-group-containing compound or a derivative thereof as and/or in the manufacture of a medicament for increasing the sensitivity of a bacterium to an antibiotic.
Preferably, the mercapto-containing compound is at least one of 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranethiol, 3-thio-1-hexanol and 1,4-butanediol bis (mercaptoacetate); further at least one of 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol and 3-thio-1-hexanol; further, at least one of 2-pyrazinylethylthiol and 2-methyl-3-tetrahydrofuranthiol is used.
2-pyrazinylethylthiol having the formula C 6 H 8 N 2 S, the CAS number is 35250-53-4, and the structural formula is shown as the formula (I).
The molecular formula of the 2-methyl-3-tetrahydrofuran thiol is C 5 H 10 OS, CAS number 57124-87-5, the structural formula is shown as formula (II).
The molecular formula of the 3-sulfenyl-1-hexanol is C 7 H 16 OS, CAS number is 51755-83-0, the constitutional formula is shown as formula (III).
1,4-butanediol bis (mercaptoacetate) has the molecular formula C 8 H 14 O 4 S 2 The CAS number is 10193-95-0, and the structural formula is shown as the formula (IV).
Preferably, the derivative includes a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer or prodrug of the thiol-containing compound.
Preferably, the bacterium is a drug-resistant bacterium that expresses a metallo beta-lactamase and/or a serine beta-lactamase; further preferably, the bacterium is at least one of Escherichia coli (Escherichia coli), klebsiella pneumoniae (Klebsiella pneumoniae), and Pseudomonas aeruginosa (Pseudomonas aeruginosa).
Preferably, the antibiotic is a β -lactam antibiotic; further preferably, the antibiotic is at least one of penicillin antibiotics, cephalosporin antibiotics, cephamycins antibiotics, thiomycins antibiotics and carbapenems antibiotics; still further preferably, the antibiotic is at least one of meropenem, imipenem, ertapenem, cephalexin, cefuroxime, cefdinir, ceftriaxone, ceftazidime, ampicillin and amoxicillin.
Preferably, the metallo-beta-lactamase is at least one of an IMP-7 type metallo-beta-lactamase, an NDM-1 type metallo-beta-lactamase and a VIM-2 type metallo-beta-lactamase; further preferably, the metallo-beta-lactamase is at least one of an IMP-7 type metallo-beta-lactamase and an NDM-1 type metallo-beta-lactamase; still further preferably, the metallo-beta-lactamase is an NDM-type 1 metallo-beta-lactamase.
Preferably, the serine beta-lactamase is a KPC-2 type serine beta-lactamase.
In a third aspect of the invention, there is provided the use of an antibiotic and a thiol-group containing compound and/or a derivative thereof in the manufacture of a medicament for inhibiting bacteria.
Preferably, the mercapto-containing compound is at least one of 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol, and 1,4-butanediol bis (mercaptoacetate); further at least one of 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol and 3-thio-1-hexanol; further, at least one of 2-methyl-3-tetrahydrofurothiol and 3-thio-1-hexanol is used.
2-pyrazinylethylthiol having the formula C 6 H 8 N 2 S, the CAS number is 35250-53-4, and the structural formula is shown as the formula (I).
The molecular formula of the 2-methyl-3-tetrahydrofuran thiol is C 5 H 10 OS, CAS number 57124-87-5, the structural formula is shown as formula (II).
The molecular formula of the 3-sulfenyl-1-hexanol is C 7 H 16 OS, CAS number is 51755-83-0, the constitutional formula is shown as formula (III).
1,4-butanediol bis (mercaptoacetate) has the molecular formula C 8 H 14 O 4 S 2 The CAS number is 10193-95-0, and the structural formula is shown as the formula (IV).
Preferably, the derivative includes a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer or prodrug of the thiol-containing compound.
Preferably, the antibiotic is a β -lactam antibiotic; further preferably, the antibiotic is at least one of penicillin antibiotics, cephalosporin antibiotics, cephamycins antibiotics, thiomycins antibiotics and carbapenems antibiotics; still further preferably, the antibiotic is at least one of meropenem, imipenem, ertapenem, cephalexin, cefuroxime, cefdinir, ceftriaxone, ceftazidime, ampicillin and amoxicillin.
Preferably, the bacterium is a drug-resistant bacterium that expresses a metallo beta-lactamase and/or a serine beta-lactamase; further preferably, the bacterium is at least one of Escherichia coli (Escherichia coli), klebsiella pneumoniae (Klebsiella pneumoniae), and Pseudomonas aeruginosa (Pseudomonas aeruginosa).
Preferably, the metallo-beta-lactamase is at least one of a metallo-beta-lactamase of the type IMP-7, a metallo-beta-lactamase of the type NDM-1 and a metallo-beta-lactamase of the type VIM-2; further preferably, the metallo-beta-lactamase is at least one of an IMP-7 type metallo-beta-lactamase and an NDM-1 type metallo-beta-lactamase; still further preferably, the metallo-beta-lactamase is an NDM-type 1 metallo-beta-lactamase.
Preferably, the serine beta-lactamase is a KPC-2 type serine beta-lactamase.
In a fourth aspect of the invention, there is provided a medicament comprising:
(1) (ii) an antibiotic; and
(2) A mercapto compound and/or a derivative thereof.
Preferably, the mercapto-containing compound is at least one of 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol, and 1,4-butanediol bis (mercaptoacetate); further at least one of 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol and 3-thio-1-hexanol; further, at least one of 2-pyrazinylethylthiol and 2-methyl-3-tetrahydrofuranthiol is used.
2-pyrazinylethylthiol having the formula C 6 H 8 N 2 S, the CAS number is 35250-53-4, and the structural formula is shown as the formula (I).
The molecular formula of the 2-methyl-3-tetrahydrofuran thiol is C 5 H 10 OS, CAS number 57124-87-5, the structural formula is shown as formula (II).
The molecular formula of the 3-sulfenyl-1-hexanol is C 7 H 16 OS, CAS number 51755-83-0, structural formula is as formula (III)) As shown.
1,4-butanediol bis (mercaptoacetate) has the molecular formula C 8 H 14 O 4 S 2 The CAS number is 10193-95-0, and the structural formula is shown as the formula (IV).
Preferably, the derivative includes a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer or prodrug of the thiol-containing compound.
Preferably, the antibiotic is a β -lactam antibiotic; further preferably, the antibiotic is at least one of penicillin antibiotics, cephalosporin antibiotics, cephamycins antibiotics, thiomycins antibiotics and carbapenems antibiotics; still further preferably, the antibiotic is at least one of meropenem, imipenem, ertapenem, cephalexin, cefuroxime, cefdinir, ceftriaxone, ceftazidime, ampicillin and amoxicillin.
Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.
Preferably, the adjuvant comprises at least one of diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, surfactant, adsorption carrier, and lubricant.
Preferably, the formulation types of the drug include solid formulations, liquid formulations, and semi-solid formulations.
Preferably, the solid formulation includes tablets, granules, powders and capsules.
Preferably, the liquid formulation comprises an injection.
Preferably, the semi-solid formulation comprises an ointment and a cream.
The invention has the beneficial effects that:
the invention discloses the application of sulfhydryl-containing compounds (2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol and 1,4-butanediol bis (thioglycolate)) or derivatives thereof in serving as and/or preparing a metallo-beta-lactamase inhibitor for the first time, wherein the sulfhydryl-containing compounds (2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol and 1,4-butanediol bis (thioglycolate)) or derivatives thereof have good inhibitory effect on beta-lactamase (metallo-beta-lactamase and/or serine beta-lactamase), can protect antibiotics from being degraded by bacteria, improve the sensitivity of the bacteria to the antibiotics and reverse the drug resistance of the bacteria to the antibiotics; meanwhile, the sulfhydryl-containing compound (2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuryl mercaptan, 3-thio-1-hexanol) and/or the derivative thereof has good synergistic effect when being combined with antibiotics, and can be used as a compound medicament for inhibiting bacteria.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The materials, reagents and the like used in the present examples are commercially available reagents and materials unless otherwise specified.
EXAMPLE 1 measurement of inhibitory Activity of thiol-group-containing Compound on beta-lactamase
The substrate is hydrolyzed by the enzyme to cause the decrease of the absorbance value, so that the degree of hydrolysis of the substrate can be characterized by the change of the absorbance, thereby judging the activity of the enzyme. The change in absorbance of the substrate after hydrolysis by metallo-beta-lactamase was measured at a wavelength of 300nm using meropenem (50. Mu.M) as a reporter substrate. The metallo-beta-lactamase comprises NDM-1, VIM-2 and IMP-7 at final concentrations of 2nM, 4nM and 5nM, respectively, 50mM HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) as buffer, and ZnSO 4 (final concentration 0.1 mM), triton X-100 (final concentration 0.01% (v/v)), bovine serum albumin (BSA, final concentration 0.1. Mu.g/mL), pH =7.2, reaction temperature 25 ℃, specific experimental methods are as follows:
1. determination of inhibitory Activity of thiol-containing Compound on IMP-7 type metallo beta-lactamase
(1) 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol and 1,4-butanediol bis (thioglycolate), respectively (captopril was added to the control group, purchased from Shanghai leaf Biotech Co., ltd., batch No. S30916), were dissolved in HEPES buffer and formulated to different concentrations (0.1, 0.5, 1, 5, 10, 50, 100, 300, 500. Mu.M, respectively), three duplicate wells were set for each concentration, 10. Mu.L of a metallo beta-lactamase solution of type IMP-7 (final concentration of 5 nM) was added, and incubation was carried out at 25 ℃ for 15min to bind the 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 2, 3-thio-1-hexanol and 1,4-butanediol bis (thioglycolate) and captopril sufficiently to the enzyme.
(2) The system is transferred into a quartz cuvette, 50 mu L of meropenem (the final concentration is 50 mu M) is added, the change of the absorbance value is rapidly measured, and the data is recorded.
(3) Calculating the inhibition rate of different concentrations of 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuryl mercaptan, 3-thio-1-hexanol, 1,4-butanediol bis (mercaptoacetate) and captopril on IMP-7 type metallo-beta-lactamase, plotting the concentration of the compound on the residual activity of IMP-7 type metallo-beta-lactamase, and calculating IC by fitting a curve 50 Values, and Ki = IC 50 /(1+[S]Km) (wherein, [ S ]]Km is the mie constant of the enzyme) to obtain Ki values.
2. Determination of inhibitory Activity of thiol-containing Compound on NDM-1 type metallo beta-lactamase
(1) Separately, 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranethiol, 3-thio-1-hexanol and 1,4-butanediol bis (thioglycolate) (control group added captopril, purchased from Shanghai Ye Biotech Co., ltd., lot No. S30916) were dissolved in HEPES buffer solutions and prepared to various concentrations (0.1, 0.5, 1, 5, 10, 50, 100, 300, 500. Mu.M, respectively), three wells were set for each concentration, 10. Mu.L of NDM-1 type metallo beta-lactamase solution (final concentration of 2 nM) was added, and incubation was carried out at 25 ℃ for 15min to allow 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranethiol, 2, 3-thio-1-hexanol, 1,4-butanediol bis (thioglycolate) and captopril to bind well to the enzyme.
(2) The system is transferred into a quartz cuvette, 50 mu L of meropenem (the final concentration is 50 mu M) is added, the change of the absorbance value is rapidly measured, and the data is recorded.
(3) Calculating the inhibition rate of different concentrations of 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuryl mercaptan, 3-thio-1-hexanol, 1,4-butanediol bis (mercaptoacetate) and captopril on NDM-1 type metallo-beta-lactamase, plotting the concentration of the compound on the residual activity of NDM-1 type metallo-beta-lactamase, and calculating IC by fitting a curve 50 The value is calculated, and the Ki value is obtained.
3. Determination of inhibitory Activity of thiol-containing Compound on VIM-2 type metallo beta-lactamase
(1) 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranethiol, 3-thio-1-hexanol and 1,4-butanediol bis (thioglycolate) (control added captopril, purchased from Shanghai leaf Biotechnology Co., ltd., lot No. S30916) were dissolved in HEPES buffer solutions and formulated to various concentrations (0.1, 0.5, 1, 5, 10, 50, 100, 300, 500. Mu.M, respectively) with three complex wells, 10. Mu.L of VIM-2 metallo beta-lactamase solution (final concentration of 4 nM) was added, and incubation was carried out at 25 ℃ for 15min to allow the enzymes to bind well to 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranethiol, 3-thio-1-hexanol and 1,4-butanediol bis (thioglycolate) (captopril).
(2) The system is transferred into a quartz cuvette, 50 mu L of meropenem (the final concentration is 50 mu M) is added, the change of the absorbance value is rapidly measured, and the data is recorded.
(3) Calculating the inhibition rate of different concentrations of 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol, 1,4-butanediol bis (mercaptoacetate) and captopril on VIM-2 metallo-beta-lactamase, plotting the concentration of the compound on the residual activity of VIM-2 metallo-beta-lactamase, and calculating IC by fitting a curve 50 The value is calculated, and the Ki value is obtained.
The results of the inhibitory activity of the mercapto-containing compounds (2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol and 1,4-butanediol bis (mercaptoacetate)) and captopril against metallic beta-lactamases of the IMP-7, NDM-1 and VIM-2 types are shown in Table 1: 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranyl thiol and 1,4-butanediol bis (mercaptoacetate) have an inhibitory effect on NDM-1 type metallo-beta-lactamase, 2-pyrazinylethylthiol and 2-methyl-3-tetrahydrofuranyl thiol have an inhibitory effect on VIM-2 type metallo-beta-lactamase, 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranyl thiol and 3-thio-1-hexanol have a good inhibitory effect on IMP-7 type metallo-beta-lactamase, and especially 1,4-butanediol bis (mercaptoacetate) has an inhibitory effect on NDM-1 type metallo-beta-lactamase better than captopril.
TABLE 1 sulfhydryl-containing Compounds and IC's of captopril against metallic beta-lactamases of IMP-7 type, NDM-1 type and VIM-2 type 50 (. Mu.M) and Ki (. Mu.M)
Figure BDA0003101274780000081
Example 2 evaluation of Effect of Using a mercapto Compound in combination with Meropenem in inhibiting production of beta-lactamase-resistant bacteria
The Minimum Inhibitory Concentration (MIC) of the combination of the sulfhydryl-containing compound and meropenem on beta-lactamase-resistant strains is determined by adopting a trace broth dilution method. The E.coli BL21 (DE 3)/pMAL-c 5x-IMP-7 used for the experiments were purchased from Shanghai Biotech Co., ltd, and E.coli BL21 (DE 3)/pET 24a-VIM-2 and E.coli BL21 (DE 3)/pET 26b-NDM-1 were obtained from gifts from professor Yang Kewu, northwest university, in the literature: zhang Yuejuan, characterization and inhibition Studies of antibiotic-resistant target protein metallo-beta-lactamase and its resistant bacteria [ D]Northwest university, 2019; also included is the clinical isolate E.coli BAA-2452 (bla) purchased from Beijing Baiohobwei Biotechnology Ltd NDM-1 ) Coli BAA-2340 (bla) KPC-2 )。
FICI is used to judge the interaction of 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 Wherein MIC A And MIC B MIC values for Compounds A and B, respectively, alone, and C A And C B Is the drug concentration of compound a and B in the effective combination. If the FICI is less than or equal to 0.5, the two medicines are considered to have synergistic action, if the FICI is more than 0.5 and less than or equal to 4, the two medicines are considered to have weak synergistic action or no related action, and if the FICI is more than or equal to 4, the two medicines are considered to have synergistic actionAntagonism is obtained. The smaller the FICI, the stronger the drug synergy.
The specific experimental method is as follows:
1. evaluation of Effect of combination of thiol-containing Compound and meropenem in inhibiting production of IMP-7 type drug-resistant bacteria
(1) Inoculating strain (E.coli BL21 (DE 3)/pMAL-c 5x-IMP-7 strain) preserved at ultralow temperature into sterile LB solid medium under aseptic operation condition, culturing overnight in 37 deg.C constant temperature incubator, selecting single colony, transferring into 3mL LB liquid medium (containing 50mg/mL ampicillin), culturing in 37 deg.C constant temperature incubator to logarithmic growth phase to obtain bacterial suspension, adjusting bacterial concentration to 0.5 McLeod concentration with McLeod turbidimeter, diluting LB liquid medium by 100 times, and counting bacteria about 1 × 10 6 CFU/mL。
(2) Adding 100 mu L of LB liquid culture medium into the 2 nd to 12 th rows of a 96-well plate, adding 100 mu L of meropenem solution (256 mu g/mL) or an inhibitor (a sulfhydryl-containing compound: 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis (thioglycolate) into the 1 st row), sucking 100 mu L of the mixed solution in the 2 nd row, adding the mixed solution into the 3 rd row, mixing the solution again, and sequentially diluting the solution by a double-ratio dilution method to obtain the drug concentration of 0.0625-128 mu g/mL; the MIC of Meropenem or a mercapto compound (2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis (mercaptoacetate)) alone against IMP-7-type drug-resistant bacteria was determined by adding 100. Mu.L of diluted bacteria per well, with three wells per concentration.
(3) Jointly diluting the liquid medicine on a 96-well plate according to the horizontal direction and the vertical direction, and performing gradient dilution on meropenem in the horizontal row, wherein the method is the same as the step (2), but the volumes of the added LB liquid culture medium and the meropenem are both 50 mu L (the final concentration of the meropenem is 0.0625-128 mu g/mL), and 50 mu L of inhibitors (a sulfhydryl-containing compound: 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranethiol, 3-sulfenyl-1-hexanol or 1,4-butanediol bis (thioglycolate)) which are diluted in multiple times are added in the vertical row, and the final concentration is 2-128 mu g/mL; each well was filled with 100. Mu.L of diluted bacterial solution, and the MIC of meropenem in combination with an inhibitor (a mercapto compound: 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis (mercaptoacetate)) against IMP-7-type drug-resistant bacteria was determined, three wells per concentration.
(4) Each set of experiments was set up with three parallel controls: taking escherichia coli ATCC25922 as a quality control standard, taking captopril as a positive control, and setting a sterile hole and a medicine-free hole at the same time; the 96-well plate was incubated in a 37 ℃ incubator for 24 hours, and the results were observed and MIC values were recorded.
2. Evaluation of effect of combination of sulfhydryl-containing compound and meropenem on inhibiting generation of VIM-2 type drug-resistant bacteria
(1) Inoculating a strain (E.coli BL21 (DE 3)/pET 24 a-VIM-2) preserved at ultralow temperature into a sterile LB solid culture medium under the aseptic operation condition, putting the strain into a constant-temperature incubator at 37 ℃ for overnight culture, selecting a single colony, transferring the single colony into 3mL of LB liquid culture medium (containing 50mg/mL of kanamycin), and culturing the single colony in the constant-temperature incubator at 37 ℃ until the logarithmic growth phase to obtain a bacterial suspension; adjusting the concentration of the bacterial liquid to 0.5 McLeod concentration by a McLeod turbidimeter, diluting the LB liquid culture medium by 100 times, and obtaining the bacterial number of about 1 × 10 6 CFU/mL。
(2) Adding 100 mu L of LB liquid culture medium into the 2 nd to 12 th rows of a 96-well plate, adding 100 mu L of meropenem solution (256 mu g/mL) or an inhibitor (a sulfhydryl-containing compound: 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis (thioglycolate), 256 mu g/mL) into the 1 st row, sucking 100 mu L of the solution in the 2 nd row after fully mixing the solution, adding the solution into the 3 rd row for mixing again, and sequentially diluting the solution by a double dilution method to obtain the drug concentration of 0.0625 to 128 mu g/mL; the MIC of Meropenem or a mercapto compound (2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis (mercaptoacetate)) alone against drug-resistant bacteria of type VIM-2 was determined by adding 100. Mu.L of diluted bacteria per well, with three duplicate wells per concentration.
(3) Jointly diluting the liquid medicine on a 96-well plate according to the horizontal direction and the vertical direction, and performing gradient dilution on meropenem in the horizontal row, wherein the method is the same as the step (2), but the volumes of the added LB liquid culture medium and the meropenem are both 50 mu L (the final concentration of the meropenem is 0.0625-128 mu g/mL), and 50 mu L of inhibitors (a sulfhydryl-containing compound: 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranethiol, 3-sulfenyl-1-hexanol or 1,4-butanediol bis (thioglycolate)) which are diluted in multiple times are added in the vertical row, and the final concentration is 2-128 mu g/mL; each well was filled with 100. Mu.L of diluted bacterial solution, and the MIC of meropenem in combination with an inhibitor (a mercapto compound: 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis (mercaptoacetate)) against drug-resistant bacteria of type VIM-2 was determined, three wells per concentration.
(4) Each set of experiments was set up with three parallel controls: taking Escherichia coli ATCC25922 as a quality control standard, taking captopril as a positive control, and simultaneously arranging a sterile hole and a drug-free hole; the 96-well plate was incubated in a 37 ℃ incubator for 24 hours, and the results were observed and MIC values were recorded.
3. Evaluation of effect of combination of sulfhydryl-containing compound and meropenem for inhibiting NDM-1-producing type drug-resistant bacteria
(1) Inoculating a strain (E.coli BL21 (DE 3)/pET 26b-NDM-1 strain) preserved at ultralow temperature into a sterile LB solid culture medium under the aseptic operation condition, putting the strain into a constant-temperature incubator at 37 ℃ for overnight culture, selecting a single colony, transferring the single colony into 3mL of LB liquid culture medium (containing 50mg/mL of kanamycin), and culturing the single colony in the constant-temperature incubator at 37 ℃ until the logarithmic growth phase to obtain a bacterial suspension; adjusting the concentration of the bacterial liquid to 0.5 McLeod concentration by a McLeod turbidimeter, diluting the LB liquid culture medium by 100 times, and obtaining the bacterial number of about 1 × 10 6 CFU/mL。
(2) Adding 100 mu L of LB liquid culture medium into the 2 nd to 12 th rows of a 96-well plate, adding 100 mu L of meropenem solution (256 mu g/mL) or an inhibitor (a sulfhydryl-containing compound: 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis (thioglycolate), 256 mu g/mL) into the 1 st row, sucking 100 mu L of the solution in the 2 nd row after fully mixing the solution, adding the solution into the 3 rd row for mixing again, and sequentially diluting the solution by a double dilution method to obtain the drug concentration of 0.0625 to 128 mu g/mL; the MIC of Meropenem or a mercapto compound (2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis (mercaptoacetate)) alone against NDM-1 type-resistant bacteria was determined by adding 100. Mu.L of diluted bacteria per well, with three wells per concentration.
(3) Jointly diluting the liquid medicine on a 96-well plate according to the horizontal direction and the vertical direction, and performing gradient dilution on meropenem in the horizontal row, wherein the method is the same as the step (2), but the volumes of the added LB liquid culture medium and the meropenem are both 50 mu L (the final concentration of the meropenem is 0.0625-128 mu g/mL), and 50 mu L of inhibitors (a sulfhydryl-containing compound: 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranethiol, 3-sulfenyl-1-hexanol or 1,4-butanediol bis (thioglycolate)) which are diluted in multiple times are added in the vertical row, and the final concentration is 2-128 mu g/mL; each well was filled with 100. Mu.L of diluted bacterial solution, and the MIC of meropenem in combination with an inhibitor (a mercapto compound: 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis (mercaptoacetate)) against NDM-1-type drug-resistant bacteria was determined, three wells per concentration.
(4) Each set of experiments was set up with three parallel controls: taking escherichia coli ATCC25922 as a quality control standard, taking captopril as a positive control, and setting a sterile hole and a medicine-free hole at the same time; the 96-well plate was incubated in a 37 ℃ incubator for 24 hours, and the results were observed and MIC values were recorded.
4. Clinical isolate e.coli BAA-2452 (bla) was inhibited by combination of thiol-containing compound and meropenem NDM-1 ) Evaluation of Effect
(1) A strain (clinical isolate E.coli BAA-2452 (bla) was stored at ultra-low temperature under aseptic conditions NDM-1 ) Inoculating the strain into a sterile LB solid culture medium, placing the strain into a 37 ℃ constant temperature incubator for overnight culture, selecting a single colony, transferring the single colony into 3mL of LB liquid culture medium, and culturing the single colony in the 37 ℃ constant temperature incubator until the logarithmic phase to obtain a bacterial suspension; adjusting the concentration of the bacterial liquid to 0.5 McLeod concentration by a McLeod turbidimeter, diluting the LB liquid culture medium by 100 times, and obtaining the bacterial number of about 1 × 10 6 CFU/mL。
(2) Add 100. Mu.L of LB liquid medium to columns 2-12 of a 96-well plate, and add 100. Mu.L of meropenem solution (256. Mu.g/mL) or inhibitor (thiol-containing compound: 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or1,4-butanediol bis (thioglycolate), 256 μ g/mL), mixing the 2 nd column liquid medicine, sucking 100 μ L, adding into the 3 rd column, mixing, diluting the liquid medicine by the multiple dilution method to obtain the medicine concentration of 0.0625-128 μ g/mL; clinical isolates E.coli BAA-2452 (bla) were assayed by adding 100. Mu.L of diluted bacterial suspension to each well and using meropenem or a thiol-containing compound (2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis (mercaptoacetate)) alone NDM-1 ) Three replicate wells per concentration.
(3) The method is the same as the step (2), but the volumes of the added LB liquid culture medium and meropenem are both 50 mu L (the final concentration of meropenem is 0.0625-128 mu g/mL), 50 mu L of inhibitor (a sulfhydryl-containing compound: 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuran thiol, 3-thio-1-hexanol or 1,4-butanediol bis (thioglycolate)) with different concentrations diluted in time is added in the column, 100 mu L of diluted bacteria liquid is added in each hole, and the clinical isolation strain E.coli (BAbla-2 (bla-2) is combined and determined by combining the meropenem with the inhibitor (a sulfhydryl-containing compound: 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuran thiol, 3-thio-1-hexanol or 1,4-butanediol bis (thioglycolate)) NDM-1 ) Three replicate wells per concentration.
(4) Each set of experiments was set up with three parallel controls: taking Escherichia coli ATCC25922 as a quality control standard, taking captopril as a positive control, and simultaneously arranging a sterile hole and a drug-free hole; the 96-well plate was placed in a 37 ℃ incubator for 24h, the results were observed and MIC values were recorded.
5. Clinical isolate e.coli BAA-2340 (bla) was inhibited by the combination of thiol-containing compounds with meropenem KPC-2 ) Evaluation of Effect
(1) The strain that was stored at ultra-low temperature (clinical isolate E. Coli BAA-2340 (bla) was used under aseptic conditions KPC-2 ) Inoculating in sterile LB solid medium, culturing overnight in 37 deg.C constant temperature incubator, selecting single colony, transferring to 3mL LB liquid medium, culturing in 37 deg.C constant temperature incubator to logarithmic growthObtaining bacterial suspension for a long time; adjusting the concentration of the bacterial liquid to 0.5 McLeod concentration by a McLeod turbidimeter, diluting the LB liquid culture medium by 100 times, and obtaining the bacterial number of about 1 × 10 6 CFU/mL。
(2) Adding 100 mu L of LB liquid culture medium into the 2 nd to 12 th rows of a 96-well plate, adding 100 mu L of meropenem solution (256 mu g/mL) or an inhibitor (a sulfhydryl-containing compound: 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol or 1,4-butanediol bis (thioglycolate) into the 1 st row), sucking 100 mu L of the mixed solution in the 2 nd row, adding the mixed solution into the 3 rd row, mixing the solution again, and sequentially diluting the solution by a double-ratio dilution method to obtain the drug concentration of 0.0625-128 mu g/mL; to each well was added 100. Mu.L of diluted bacterial solution, and the clinical isolate E.coli BAA-2340 (bla acetate) was assayed using meropenem or a mercapto compound (2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranethiol, 3-thio-1-hexanol or 1,4-butanediol bis (mercaptoacetate)) alone KPC-2 ) Three replicate wells per concentration.
(3) The same procedure as in step (2) was followed by jointly diluting the drug solution in both the lateral and longitudinal directions on a 96-well plate in a gradient manner of meropenem in the lateral row, except that the volumes of the LB liquid medium and meropenem added were 50. Mu.L each (the final concentration of meropenem was 0.0625 to 128. Mu.g/mL), 50. Mu.L of an inhibitor (a mercapto compound: 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranethiol, 3-thio-1-hexanol or 1,4-butanediol bis (thioglycolate) diluted in multiple proportions in advance and in a final concentration of 2 to 128. Mu.g/mL) was added in the longitudinal row, and 100. Mu.L of the diluted bacterial solution was added to each well to measure meropenem and the inhibitor (a mercapto compound: 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranethiol, 3-thio-1-hexanol or 1,4-butanediol bis (thioglycolate)) to be used in combination with a clinically isolated strain E.coli-2340 KPC-2 ) Three replicate wells per concentration.
(4) Each set of experiments was set up with three parallel controls: taking Escherichia coli ATCC25922 as a quality control standard, taking captopril as a positive control, and simultaneously arranging a sterile hole and a drug-free hole; the 96-well plate was incubated in a 37 ℃ incubator for 24 hours, and the results were observed and MIC values were recorded.
The results of the antibacterial activity of meropenem in combination with a mercapto-containing compound (2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol and 1,4-butanediol bis (mercaptoacetate)) against drug-resistant bacteria expressing NDM-1, VIM-2, IMP-7 or KPC-2 are shown in Table 2: the inhibitors (mercapto compound (2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol, 1,4-butanediol bis (thioglycolate)), captopril) can improve the antibacterial activity of meropenem: when the concentration of the inhibitor is 128 mug/mL, the inhibitor is used together with meropenem, the bacteriostatic effect of the meropenem on various drug-resistant bacteria expressing beta-lactamase can be improved, and compared with the single use of the meropenem, the combined use can effectively reduce the MIC value of the meropenem on drug-resistant strains, and can be reduced by 8 times at most.
FICI values for the combination of thiol-containing compounds (2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol and 1,4-butanediol bis (thioglycolate)) or captopril with meropenem for beta-lactamase expressing resistant bacteria are shown in Table 3: the FICI value of the combination of the sulfhydryl-containing compound (2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-sulfenyl-1-hexanol) and meropenem on drug-resistant bacteria expressing NDM-1 type metallo-beta-lactamase is less than or equal to 0.5, which shows that the sulfhydryl-containing compound and the meropenem have good synergistic effect; the FICI value of the combination of 2-pyrazinylethylthiol and meropenem on drug-resistant bacteria expressing IMP-7 type metallo beta-lactamase is less than or equal to 0.5, which shows that the two have good synergistic effect; the FICI value of the combination of 2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol and meropenem on drug-resistant bacteria expressing KPC-2 type serine beta-lactamase is less than or equal to 0.5, which shows that the combination has good synergistic effect, and the FICI value of the combination of captopril and meropenem on drug-resistant bacteria expressing KPC-2 type serine beta-lactamase is more than 1, which shows that the combination does not have synergistic effect.
The above results show that: when the composition is used with meropenem, a sulfhydryl-containing compound (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol) has effective synergistic antibacterial activity on drug-resistant bacteria expressing NDM-1 type metallo-beta-lactamase, 2-pyrazinylethanethiol has effective synergistic antibacterial activity on drug-resistant bacteria expressing IMP-7 type metallo-beta-lactamase, and a sulfhydryl-containing compound (2-pyrazinylethanethiol, 2-methyl-3-tetrahydrofuranthiol) has effective synergistic antibacterial activity on drug-resistant bacteria expressing KPC-2 type serine beta-lactamase, shows that the sulfhydryl-containing compound (2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol) can be used as an NDM-1 type metallo beta-lactamase inhibitor, 2-pyrazinylethylthiol can be used as an IMP-7 type metallo beta-lactamase inhibitor, and the sulfhydryl-containing compound (2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol) can be used as a KPC-2 type serine beta-lactamase inhibitor, can reverse the drug resistance of carbapenem resistant bacteria, effectively protects meropenem from being hydrolyzed by NDM-1 type, IMP-7 type and KPC-2 type metallo beta-lactamase, improves the production of NDM-1 type by the meropenem, the antibiotic activity of IMP-7 type and KPC-2 type metallo beta-lactamase. Therefore, the sulfhydryl-containing compound (2-pyrazinylethylthiol, 2-methyl-3-tetrahydrofuranthiol, 3-thio-1-hexanol) can be used as a beta-lactamase inhibitor to prepare a compound preparation with beta-lactam antibiotics.
TABLE 2 MIC (μ g/mL) of thiol-containing Compounds or Captopril in combination with Meropenem for drug-resistant bacteria expressing β -lactamase
Figure BDA0003101274780000131
Figure BDA0003101274780000141
TABLE 3 synergistic antimicrobial index (FICI) of combinations of thiol-containing compounds or captopril with meropenem against beta-lactamase-expressing drug-resistant bacteria
Figure BDA0003101274780000142
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (4)

1. Use of a thiol-containing compound which is 2-pyrazinylethylthiol or a pharmaceutically acceptable salt thereof for the preparation of a beta-lactamase inhibitor;
the beta-lactamase is a metallo beta-lactamase;
the metallo-beta-lactamase is at least one of IMP-7 type metallo-beta-lactamase, NDM-1 type metallo-beta-lactamase and VIM-2 type metallo-beta-lactamase.
2. Use of a thiol-containing compound which is 2-pyrazinylethylthiol or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for increasing the sensitivity of bacteria to antibiotics;
the antibiotic is beta-lactam antibiotic;
the bacteria are drug-resistant bacteria expressing metallo-beta-lactamase and/or serine beta-lactamase;
the metallo-beta-lactamase is at least one of IMP-7 type metallo-beta-lactamase and NDM-1 type metallo-beta-lactamase;
the serine beta-lactamase is KPC-2 type serine beta-lactamase.
3. Use of an antibiotic and a thiol-containing compound which is 2-pyrazinylethylthiol and/or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for inhibiting bacteria;
the antibiotic is beta-lactam antibiotic;
the bacteria are drug-resistant bacteria expressing metallo-beta-lactamase and/or serine beta-lactamase;
the metallo-beta-lactamase is at least one of IMP-7 type metallo-beta-lactamase and NDM-1 type metallo-beta-lactamase;
the serine beta-lactamase is KPC-2 type serine beta-lactamase.
4. A medicine for inhibiting bacteria comprises the following components:
(1) (ii) an antibiotic; and
(2) A mercapto-containing compound and/or a pharmaceutically acceptable salt thereof;
the sulfhydryl-containing compound is 2-pyrazinylethylthiol;
the antibiotic is beta-lactam antibiotic;
the bacteria are drug-resistant bacteria expressing metallo-beta-lactamase and/or serine beta-lactamase;
the metallo-beta-lactamase is at least one of IMP-7 type metallo-beta-lactamase and NDM-1 type metallo-beta-lactamase;
the serine beta-lactamase is KPC-2 type serine beta-lactamase.
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"Principles and current strategies targeting metallo‐β‐lactamase mediated antibacterial resistance";Yu-Hang Yan et al;《Medicinal Research Reviews》;20200225;第40卷(第5期);第1558-1592页 *

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