Method for inhibiting klebsiella pneumoniae carbapenemase-2

Abstract

There is provided a method of at least partially inhibiting the function of Klebsiella pneumoniae carbapenemase-2 (KPC-2) or a variant thereof, the method comprising reacting said KPC-2 with a compound selected from the group consisting of 5-chloro-1, 3-dihydro-1-hydroxy-2, 1-benzoxaborolane, 5-fluoro-1, 3-dihydro-1-hydroxy-2, 1-benzoxaborolane, 5-fluoro-1, 3-dihydro-1-hydroxy-2, 1-benzoxaborolane, 5-fluoro-1, 3-dihydro-1-hydroxy-2, 1-benzoxaborolane, 5-chloro-1, 3-dihydro-1-hydroxy-2, 1-benzoxaborolane, 5-chloro-1, 3-dihydro-1-hydroxy-2, 1- The present invention relates to a method for contacting a beta-lactam antibiotic-resistant bacterium with a benzoxaborolane selected from the group consisting of 1, 1-benzoxaborolane and mixtures thereof, which can be used to treat an infection with a beta-lactam antibiotic-resistant bacterium in a subject.

Description

Technical Field

[0001] The present disclosure relates to methods of inhibiting Klebsiella pneumoniae carbapenemase-2, which can be used to treat infections with β-lactam antibiotic-resistant bacteria. Background Art

[0002] Since the first discovery of antibiotics in the 19th century, humans have opened the door to fight pathogenic microorganisms. A variety of antibiotics have been discovered, such as quinolone β-lactam antibiotics, macrolides, and aminoglycosides. Due to the discovery of these compounds, human life expectancy has been extended. However, with the widespread use and abuse of antibiotics, bacteria have gradually developed resistance to them, resulting in ineffective treatment. Over time, this phenomenon has become more and more serious. In 2014, a study by Temkin et al. evaluated the frequency of Escherichia coli and Klebsiella pneumoniae that produce third-generation cephalosporins and carbapenems in 193 countries. The study showed that these bacteria have caused 6.4 million bloodstream infections and 50.1 million serious infections. In 2010, Thai hospitals found that the mortality rate caused by multidrug-resistant bacterial infections was as high as 43%. In 2015, Alessandro et al. estimated that in the EU and European Economic Area countries, drug-resistant bacteria caused 671,689 infections, including 33,110 deaths. According to statistics, the main pathogens that cause infection and even death are the following six, including Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, A. baumannii and Pseudomonas aeruginosa. According to statistics, the cost of treating each patient infected with antibiotic-resistant bacteria is $29,000. The U.S. healthcare system loses about $35 billion each year due to antibiotic resistance. It is estimated that without effective new drugs, the cost of antibiotic-resistant bacterial infections could be as high as $120 trillion.

[0003] Carbapenem antibiotics such as meropenem and imipenem are considered the last line of defense against multidrug-resistant bacteria. However, Enterobacteriaceae bacteria can hydrolyze these antibiotics by causing carbapenemase hydrolysis, rendering the drugs ineffective. Escherichia coli and Klebsiella pneumoniae are prominent representatives of carbapenem-resistant Enterobacteriaceae (CRE). Their resistance mechanism is the production of a class of enzymes that can hydrolyze carbapenem antibiotics, such as KPC, VIM, NDM, and OXA-48. Klebsiella pneumoniae carbapenemase (KPC) is the most common of the carbapenemases. It was first reported in 1996. Subsequently, this carbapenemase has spread widely among Gram-negative bacteria. Its spread between microorganisms is facilitated by horizontal gene transfer via plasmids. In a short period of time, bacteria that can produce KPC appeared in multiple countries, including the United States, Puerto Rico, Colombia, Greece, and Israel. In addition, outbreaks of bacteria containing KPC-2 have also been reported in Europe and South America. In 2007, the first case of KPC strain was reported in China. Giacobbe et al. reported that the mortality rate associated with infection with KPC-positive pathogens was as high as 51%. Faced with the severe challenge of CRE, it is necessary to quickly identify effective and safe inhibitors to reduce its threat to public health.

[0004] As a new class of antifungal drugs, 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2718) is a benzoxaborane compound that inhibits fungal growth by inhibiting protein synthesis. AN2718 is currently being developed for the topical treatment of tinea pedis, but there are few reports on its inhibition of bacterial growth. AN2718 has high safety, good drug properties, and a small molecular weight. It is easy to design and modify it based on the skeleton to produce compounds with better efficacy, and has good potential clinical application value. Summary of the invention

[0005] Provided herein is a method for at least partially inhibiting the function of Klebsiella pneumoniae carbapenemase-2 (KPC-2) or a variant thereof, the method comprising contacting KPC-2 with a benzoxaborolane selected from the group consisting of 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane, 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane, and mixtures thereof.

[0006] In certain embodiments, the benzoxaborolane is 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane.

[0007] In certain embodiments, the variant of KPC-2 is KPC-93.

[0008] In certain embodiments, the benzoxaborolane is 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane and the variant of KPC-2 is KPC-93.

[0009] In a second aspect, provided herein is a method of treating a bacterial infection in a subject in need thereof, the method comprising co-administering a therapeutically effective amount of a β-lactam antibiotic and a benzoxaborolane selected from the group consisting of 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane, 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane, and mixtures thereof, wherein the bacterial infection is caused by a bacterium expressing Klebsiella pneumoniae carbapenemase-2 (KPC-2) or a variant thereof.

[0010] In certain embodiments, the bacterium is Aeromonas caviae, Aeromonas hydrophila, Aeromonas veroni, Citrobacter amalonaticus, Citrobacter freundii, Citrobacter koseri, Citrobacter portucalensis, Citrobacter youngae, Enterobacter asburiae, Enterobacter cloacae, Enterobacter hormaechei, Enterobacter kobei, Escherichia coli, Klebsiella aerogenes, Klebsiella michiganensis, Klebsiella oxytoca, oxytoca, Klebsiella pneumoniae, Klebsiella quasipneumoniae, Morganella morganii, Proteus mirabilis, Pseudomonas aeruginosa, Raoultella planticola, Salmonella enterica, or Serratia marcescens.

[0011] In certain embodiments, the bacterium is Escherichia coli, Enterobacter cloacae, Klebsiella aerogenes, carbapenem-resistant Klebsiella pneumoniae, or hypervirulent Klebsiella pneumoniae.

[0012] In certain embodiments, the beta-lactam antibiotic is a cephalosporin, a carbapenem, a penam, or a monobactam.

[0013] In certain embodiments, the β-lactam antibiotic is a carbapenem.

[0014] In certain embodiments, the β-lactam antibiotic is a carbapenem selected from the group consisting of Biapenem, Ertapenem, Doripenem, Imipenem, Meropenem, Tebipenem, and Panipenem, or a cephamycin, such as Cefoxitin, Cefmetazole, and Cefotetan.

[0015] In certain embodiments, the carbapenem is meropenem.

[0016] In certain embodiments, the benzoxaborolane is 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane.

[0017] In certain embodiments, the bacteria is carbapenem-resistant Klebsiella pneumoniae, hypervirulent Klebsiella pneumoniae, Escherichia coli, or Enterobacter cloacae, the benzoxaborol is 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborol, and the β-lactam antibiotic is a carbapenem selected from the group consisting of biapenem, ertapenem, doripenem, imipenem, meropenem, tebipenem, and panipenem, or a cephamycin, such as cefoxitin, cefmetazole, and cefotetan.

[0018] In certain embodiments, the bacteria is carbapenem-resistant Klebsiella pneumoniae or hypervirulent Klebsiella pneumoniae, the benzoxaborol is 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborol, and the beta-lactam antibiotic is a carbapenem and is meropenem.

[0019] In certain embodiments, the variant of KPC-2 is KPC-93. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Time-kill curves are depicted for: (A) AN2718 alone; (B) meropenem alone; (C) 160 μM AN2718 in combination with meropenem; (D) 80 μM AN2718 in combination with meropenem; (E) 40 μM AN2718 in combination with meropenem; (F) 20 μM AN2718 in combination with meropenem.

[0021] Figure 2 Depicted are the results of inhibition assays of AN2718 on the enzyme activities of (A) KPC-2, and mutants of (B) KPC-93 and (C) KPC-41.

[0022] Figure 3Depicted are (A) the docking results of AN2718 and KPC-2 enzyme (cartoon form); (B) the docking results of AN2718 and KPC-2 enzyme (surface form); the dotted lines represent hydrogen bonds.

[0023] Figure 4 Experimental results of AN2718 and meropenem treatment are depicted. DETAILED DESCRIPTION

[0024] definition

[0025] Throughout this disclosure, unless the context requires otherwise, the words "comprise, comprises, comprising" or variations thereof will be understood to imply the inclusion of the stated integer or group of integers, but not the exclusion of any other integer or group of integers. It should also be noted that in this disclosure, particularly in the claims and / or paragraphs, terms such as "comprise, comprises, comprising" may have the meanings assigned to them by U.S. patent law; for example, they may mean "include, include, including"; and terms such as "consisting essentially of / consists essentially of" have the meanings assigned to them by U.S. patent law, for example, it allows the presence of elements not expressly described, but excludes elements existing in the prior art or elements that affect the basic or novel characteristics of the invention.

[0026] Furthermore, throughout the present disclosure and claims, unless the context requires otherwise, the words "include, includes, including" or variations such as these will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0027] Unless otherwise expressly stated, nouns used herein without quantifiers include one or more of the noun. In addition, if the term "about" is used before a quantitative value, the present teaching also includes the specific quantitative value itself, unless otherwise specifically stated. As used herein, unless otherwise stated or inferred, the term "about" refers to a variation of ±10%, ±7%, ±5%, ±3%, ±1%, or ±0% of the nominal value.

[0028] The term "subject" as used herein refers to an animal, typically a mammal or a human, that will be or has been the subject of treatment, observation, and / or experiment. When the term is used in conjunction with the administration of a compound or drug, the subject is the subject of treatment, observation, and / or administration of the compound or drug.

[0029] The terms "co-administration" and "co-administration" refer to both simultaneous administration (administration of two or more therapeutic agents at the same time) and non-simultaneous administration (administration of one or more additional therapeutic agents at a different time than administration of one or more therapeutic agents), so long as the therapeutic agents are present in the patient's body at the same time to some extent.

[0030] As used herein, the term "therapeutically effective amount" refers to that amount of active compound or pharmaceutical agent that elicits the biological or pharmaceutical response sought by the researcher, veterinarian, clinician or physician in cell cultures, tissue systems, animals or humans, including the alleviation of symptoms of the disease, condition or disorder being treated.

[0031] Provided herein is a method for at least partially inhibiting the function of Klebsiella pneumoniae carbapenemase-2 (KPC-2) or a variant thereof, the method comprising contacting KPC-2 with a benzoxaborolane selected from the group consisting of 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane, 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane, and mixtures thereof. The method for at least partially inhibiting the function of KPC-2 or a variant thereof can be performed in vitro or in vivo.

[0032] The present disclosure also provides a method of treating a bacterial infection in a subject in need thereof, the method comprising co-administering a therapeutically effective amount of a β-lactam antibiotic and a benzoxaborolane selected from the group consisting of 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane, 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane, and mixtures thereof, wherein the bacterial infection is caused by a bacterium expressing Klebsiella pneumoniae carbapenemase-2 (KPC-2) or a variant thereof.

[0033] Variants of KPC-2 may include one or more single amino acid substitutions and / or 2 to 7, 3 to 7, or 4 to 6 amino acid insertions relative to wild-type KPC-2 (GenBank Accession No. EU784136.1). Such variants are known to increase the catalytic efficacy of β-lactam antibiotic hydrolysis by ten-fold or more. In certain embodiments, variants of KPC-2 include one or more amino acid substitutions selected from the group consisting of P104R (KPC-5), V240F, M49I, H274Y, V240G (KPC-6), V240A, and P104L relative to KPC-2; or KPC-2 comprising amino acid insertions, such as KPC-93, which includes five amino acid insertions located between Ambler positions 267 and 268, or KPC-41, which includes three amino acid insertions located between Ambler positions 269 and 270. As Figure 2 As shown, the methods described herein are unexpectedly effective against bacteria expressing KPC-93.

[0034] The results provided below demonstrate the efficacy of the benzoxaborolanes described herein in inhibiting KPC-2 and the resulting resensitization of carbapenem-resistant Klebsiella pneumoniae to menoprem. Since the KPC-2 gene can be carried on a plasmid, which will allow the gene to be easily transferred between different bacterial strains by horizontal gene transfer, other bacteria besides Klebsiella pneumoniae have also been shown to express KPC-2.

[0035] Thus, the methods described herein can be used to treat bacterial infections caused by bacteria other than Klebsiella pneumoniae, for example, bacteria from the genus Staphylococcus, such as Staphylococcus aureus or Staphylococcus epidermidis; Streptococcus, such as Streptococcus agalactiae, Streptococcus pneumoniae, or Streptococcus faecalis; Micrococcus, such as Micrococcus luteus; Bacillus, such as Bacillus subtilis; Listerella, such as Listerella monocytogenes; other members of the genus Escherichia; Proteus, such as Proteus mirabilis; mirabilis or Proteus vulgaris; Salmonella, such as Salmonella typhosa; Shigella, such as Shigella sonnef; Enterobacter, such as Enterobacter aerogenes or Enterobacter cloacae; Serratia, such as Serratia marcescens; Pseudomonas, such as Pseudomonas aeruginosa; Acinetobacter, such as Acinetobacter baumanii; Nocardia, such as Nocardia autotrophica; autotrophica); Escherichia, such as Escherichia coli; or Mycobacterium, such as Mycobacterium fortuitum, and combinations thereof.

[0036] In certain embodiments, the bacterium is Aeromonas caviae, Aeromonas hydrophila, Aeromonas veronii, Citrobacter amalonaticus, Citrobacter freundii, Citrobacter cohnii, Citrobacter portugal, Citrobacter youngae, Enterobacter afscher, Enterobacter cloacae, Enterobacter hallii, Enterobacter kobe, Escherichia coli, Klebsiella aerogenes, Klebsiella michiganensis, Klebsiella oxytoca, Klebsiella pneumoniae, Klebsiella quasi-pneumoniae, Morganella morganii, Proteus mirabilis, Pseudomonas aeruginosa, Raoultella planticola, Salmonella enterica, or Serratia marcescens.

[0037] In certain embodiments, the bacterium is Escherichia coli, Enterobacter cloacae, Klebsiella aerogenes, carbapenem-resistant Klebsiella pneumoniae, or hypervirulent Klebsiella pneumoniae.

[0038] KPC-2 is a class A β-lactamase with a broad spectrum of β-lactam antibiotic substrates. Some examples of β-lactam antibiotics that can be used in combination with the methods of the present disclosure include, but are not limited to, β-lactams, including penicillins, carbapenems, oxopenems, penems, carbapenems, monobactams, cephems, carbacephems, or oxacephems. However, KPC-2 is known to be particularly effective in hydrolyzing carbapenems and cephamycins.

[0039] Exemplary β-lactam antibiotics include, but are not limited to, penicillins such as Benzylpenicillin (G), Benzathine Benzylpenicillin, Procaine Benzylpenicillin, Phenoxymethylpenicillin (V), Propicillin, Pheneticillin, Pzidocillin, Plometocillin, Penamecilli, Cloxacillin, Dicloxacillin, Flucloxacillin, Oxacillin, Nafcillin, Methyl Methicillin, Amoxicillin, Ampicilli, Pivampicillin, Hetacillin, Bacampicillin, Metampicillin, Talampicillin, Epicillin, Ticarcillin, Carbenicillin, Carindacillin, Timocillin Temocillin, Piperacillin, Azlocillin, Mezlocillin, Mecillinam, Pivmecillinam, and Sulbenicillin; Penems, such as Faropenem and Ritipenem; Carbapenems, such as Ertapenem, Doripenem, Imipenem, Meropenem, Biapenem, and Panipenem; Cephalosporins, such as Cefazoli 、Cefalexin、Cefadroxyproline、Cefapirin、Cefazedone、Cefazaflur、Cefradine、Cefroxadine、Ceftezole、Cefalloglycin、Cefacetrile、Cefalonium、Cefaloridine、Cefalotin、Ceftrizine, Cefaclor, Cefotetan, Cephamycin, Cefoxitin, Cefprozil, Cefuroxime, Cefuroxime axetil, Cefamandole, Cefminox, Cefonicid, Ceforanide, Cefotiam, Cefbuperazone, Cefuzonam, Cefmetazole, Carbacephem, Loracarbef, Cefixime, Ceftriaxone xon), Ceftazidime, Cefoperazone, Cefdinir, Cefcapene, Cefdaloxime, Ceftizoxime, Cefmenoxime, Cefotaxime, Cefpiramide, Cefpodoxime, Ceftibu ten), Cefditoren, Cefetamet, Cefodizime, Cefpimizole, Cefsulodin, Cefteram, Ceftiolene, Oxacephem, Flomoxef, Latamoxef, Cefepime, Cefazolin ozopran), Cefpirome, Cefquinome, Ceftarolinefosamil, Ceftolozane, Ceftobiprole, Ceftiofur, Cefquinome, and Cefovecin; and monobactams such as Aztreonam, Tigemonam, Carumonam, and Nocardicin A.

[0040] In certain embodiments, the beta-lactam antibiotic is a cephalosporin, a carbapenem, a penicillam, or a monobactam.

[0041] In certain embodiments, the beta-lactam antibiotic is a carbapenem, such as biapenem, ertapenem, doripenem, imipenem, meropenem, tebipenem, and panipenem, or a cephamycin, such as cefoxitin, cefmetazole, and cefotetan.

[0042] The compounds described herein can be administered according to treatment regimens known in the art. It will be apparent to those skilled in the art that the administration of the benzoxaborolanes and β-lactam antibiotics described herein can vary depending on the disease being treated and the known effects of the β-lactam antibiotic on the disease. In addition, based on the observed effects of the administered therapeutic agent (i.e., β-lactam antibiotic) on the patient, and based on the observed response of the disease to the administered therapeutic agent, the treatment regimen (e.g., dosage and number of administrations) can be altered according to the knowledge of an experienced clinician.

[0043] In addition, in general, the benzoxaborolanes and β-lactam antibiotics described herein need not be administered in the same pharmaceutical composition and may have to be administered by different routes due to different physical and chemical properties. For example, the benzoxaborolanes described herein may be administered intravenously to produce and maintain good blood levels, while the β-lactam antibiotic may be administered orally. The determination of the mode of administration and the appropriateness of administration in the same pharmaceutical composition, where possible, are well known to skilled clinicians. Initial administration may be performed according to established protocols known in the art, and then, based on the observed effects, the skilled clinician may modify the dosage, mode of administration, and time of administration.

[0044] The specific choice of β-lactam antibiotic will depend on the attending physician's diagnosis and his or her judgment of the patient's condition and the appropriate treatment regimen.

[0045] The benzoxaborolanes described herein and the β-lactam antibiotic can be administered simultaneously (e.g., simultaneously, substantially simultaneously, or within the same treatment regimen) or sequentially, depending on the nature of the bacterial infection, the condition of the patient, and the actual choice of β-lactam antibiotic to be administered in combination with the benzoxaborolanes described herein (i.e., in a monotherapy regimen).

[0046] If the benzoxaborolane and the β-lactam antibiotic described herein are not administered simultaneously or substantially simultaneously, the optimal order of administration of the benzoxaborolane and the β-lactam antibiotic described herein may be different for different bacterial infections. Thus, in some cases, the benzoxaborolane described herein may be administered first, followed by the β-lactam antibiotic; and in other cases, the β-lactam antibiotic may be administered first, followed by the benzoxaborolane described herein. This alternating administration may be repeated during a single treatment regimen. Determination of the order of administration and number of repeated administrations of each therapeutic agent during a treatment regimen is within the knowledge of a skilled physician after evaluating the disease being treated and the patient's condition. For example, a β-lactam antibiotic may be administered first, followed by continued treatment by administration of a benzoxaborolane described herein, followed by administration of a β-lactam antibiotic, and so on, until the treatment regimen is completed.

[0047] Thus, based on experience and knowledge, the practitioner can modify each regimen of administration of the therapeutic components (the benzoxaborolanes and the beta-lactam antibiotic described herein) according to the needs of the individual patient as treatment progresses.

[0048] Materials and methods

[0049] Strains and reagents

[0050] KPC-74T, which carries Escherichia coli containing KPC-2 plasmid, was constructed in the laboratory; Hvkp, which was obtained by laboratory isolation; AN2718, which was purchased from Target Mol Technology Co., Ltd; BBLTM Mueller Hinton II liquid culture medium was purchased from BD Company, USA; meropenem was purchased from Meilun Biotechnology Co., Ltd; LB liquid culture medium was purchased from Qingdao High tech Industrial Park Haibo Biotechnology Co., Ltd; microplate reader (UV1900i, Shimadzu Company).

[0051] Minimum inhibitory concentration determination by chessboard method

[0052] This study referred to the standards of the Clinical and Laboratory Standards Institute (CLSI) in the United States and the previous improved methods in the laboratory, and measured the fractional inhibitory concentration through checkerboard assays. First, KPC-74T was activated and inoculated into Lysogeny broth medium, and cultured overnight at 37 °C. 100 μL of MHB medium was added to each well of a sterile 96-well plate; then 100 μL of AN2718 solution was added to the first row and serially diluted along the vertical axis. Then, meropenem solution was added to the first column and serially diluted along the horizontal axis. Finally, except for the negative control, 100 μL of bacterial suspension ≈ 1×10 6 CFU mL -1 was added. After the above operations were completed, the 96-well plate was placed in an incubator at 37 °C and incubated for 18 hours. The bacterial suspensions in each well were observed under appropriate light, and the drug concentration corresponding to the well where no bacterial growth was observed was the MIC. The synergistic effect was determined by calculating the FIC. The FIC can be calculated according to Equation (1):

[0053]

[0054] MICa is the individual MIC of compound a; MICab is the MIC of the combination of compound a and compound b; MICb is the individual MIC of compound b; MICba is the MIC of the combination of compound b and compound a; FICa is the FIC of compound a; FICb is the FIC of compound b. According to the standard definition, FICI ≤ 0.5 is defined as synergism; 0.5 < FICI ≤ 1 is defined as additivity; 1 < FICI ≥ 4 is defined as indifference; FICI > 4 is defined as antagonism.

[0055] Determination of time-kill curves

[0056] The overnight-cultured bacteria KPC-74T were inoculated onto MHB medium at a ratio of 1:10000 and incubated at 37 °C and 225 rpm for 2 hours (early exponential growth phase) or 5 hours (late exponential growth phase). After incubation, the corresponding concentration of AN2718 or meropenem solution was added, and incubation was continued at 37 °C and 225 rpm. The time points were set at 0, 1, 3, 5, 7, and 24 hours. At each time point, 100 μL of the bacterial suspension was taken, centrifuged at 10000 g for 1 min, resuspended with PBS, serially diluted tenfold, and 10 μL of the liquid was taken from each dilution and dropped onto MHA plates. The plates were placed in an incubator at 37 °C for 24 hours and then taken out. Each plate was counted and the total number of bacteria was calculated. The experiment was repeated three times.

[0057] Determination of enzyme activity

[0058] Enzyme activity was measured in 50 mM phosphate buffer (pH 7.0) using a Shimadzu UV-1900i spectrophotometer to measure inhibition. The total volume of the enzyme activity reaction system was 500 μL. The reaction temperature was set to 37°C, 0.3 nM KPC-2 enzyme was pre-incubated with different concentrations of AN2718 in phosphate buffer (pH 7.0) for 5 minutes, and then 60 μL of nitrocefin was added. The inhibitory effect of AN2718 on the hydrolysis of nitrocefin was measured by detecting the change in absorbance. The 50% inhibition concentration was determined by the inhibition curve of the control activity percentage and the inhibitor concentration. The experiment was repeated three times.

[0059] Molecular docking

[0060] The AN2718 3D structure was downloaded from the PubChem database. The KPC-2 protein structure was downloaded from the PDB database PDB ID: 5LL7. First, the AN2718 and KPC-2 protein structures were preprocessed to minimize energy and remove water and add hydrogen. The active site coordinates of KPC-2 are X: 8.04, Y: 68.49, Z: 1.15. Semi-flexible docking was used for docking. The best binding conformation of AN2718 and KPC-2 protein was screened according to the size of the binding energy, and the binding conformation was analyzed and visualized using PyMoL 2.5 software.

[0061] Animal experiments

[0062] This experiment treated mice infected with the Hvkp2 strain. A total of 35 4-week-old SPF male ICR mice were selected and divided into seven groups. The seven groups were set up as saline control group, AN2718 monotherapy group (16 mg / kg), meropenem monotherapy group (16 mg / kg), AN2718 / meropenem combination group 1 (8 mg / kg + 8 mg / kg), AN2718 / meropenem combination group 2 (8 mg / kg + 16 mg / kg), AN2718 / meropenem combination group 3 (16 mg / kg + 8 mg / kg) and AN2718 / meropenem combination group 4 (8 mg / kg + 16 mg / kg). First, Hvkp2 was incubated at 37°C overnight, re-inoculated into fresh culture medium, and shaken at 37°C until OD 600 to 0.5, wash once with physiological saline and resuspend, and adjust the bacterial concentration to about 1×10 6 CFU / mL, for later use. 100 μL of bacterial suspension was injected into the peritoneal cavity of each mouse. Saline or drug was injected 1 hour after infection. 12 hours later, the second dose of drug treatment was given, for a total of 5 injections, with an interval of 12 hours. The survival status of mice in each group was observed and counted during the experiment.

[0063] Data analysis

[0064] SPSS 20.0 software was used to perform statistical analysis on the data expressed as (X ± SD). Multiple comparisons and one-way ANOVA were used to analyze and compare the data of each group. (P < 0.05 indicates a significant difference, and P < 0.01 indicates a highly significant difference).

[0065] result

[0066] Minimum inhibitory concentration results of chessboard method

[0067] The results of the chessboard test (Table 1) show that when the drug AN2718 is used alone, the MIC value for KPC-74T bacteria is 13.44μg / mL; when meropenem is used alone, the MIC value is 64μg / mL. When the two drugs are used in combination, the MIC value of AN2718 for KPC-74T decreases from 13.44μg / mL to 3.36μg / mL, and the MIC value decreases by 4 times; the MIC value of meropenem for KPC-74T decreases from 64μg / mL to 0.0625μg / mL, and the MIC value decreases by 1024 times. According to formula (1), the FICI value can be calculated to be 0.25, which is less than 0.5, indicating that the combination of the two drugs has a synergistic effect.

[0068] Table 1 MIC values ​​of AN2718 and meropenem alone and in combination

[0069]

[0070] Note: S stands for single; C stands for combination.

[0071] Time-kill curve results

[0072] from Figure 1 (A) It can be seen that among the different concentrations of AN2718 within 24 hours, the highest concentration that had no effect on the growth of KPC-74T and had no inhibitory effect was 160 μM (26.88 μg / mL); Figure 1(B) It can be seen that meropenem (2μg / mL~64μg / mL) has a certain inhibitory effect on the growth of KPC-74T in the first 5 hours. The inhibitory effect increases with the increase of concentration, but there is no significant difference in the number of colonies between the control group and WT at 24 hours. Meropenem at a concentration of 128μg / mL has a significant inhibitory effect on the growth of KPC-74T, but it still cannot kill all bacteria within 24 hours. At a concentration of 160μM, a concentration of 4μg / mL of meropenem can kill all bacteria within 24 hours; at a concentration of 80μM, a concentration of 4μg / mL of meropenem can kill most bacteria within 24 hours, with only a very small amount of bacteria remaining, and the remaining concentrations can still kill all bacteria within 24 hours. Continue to reduce the concentration of AN2718 to 40μM. Concentrations of 4μg / mL and 8μg / mL of meropenem cannot kill all bacteria within 24 hours, and the remaining concentrations can still effectively kill bacteria. When the concentration of AN2718 was 20 μM, the bactericidal efficacy of meropenem at a concentration of 128 μg / mL gradually increased within 24 hours and killed all bacteria. In summary, under the synergistic effect of AN2718, meropenem was able to restore its bactericidal ability against KPC-74T, and its bactericidal effect increased with the increase of AN2718 concentration.

[0073] Enzyme activity measurement results

[0074] from Figure 2 (A) It can be seen that the inhibitory effect of AN2718 on KPC-2 enzyme activity increases with the increase of its concentration. When the KPC-2 enzyme concentration is 0.3nM and the substrate concentration is 23.16μM, the half-inhibitory concentration IC50 of AN2718 on KPC-2 activity is measured to be 0.4985μM. It shows a reliable inhibitory effect. For KPC-93, Figure 2 As shown in (B), the half inhibition concentration IC50 is 0.1159μM. The inhibitory effect is still significant. The inhibitory effect on KPC-41 is slightly worse than the first two enzymes, with a half inhibition concentration IC50 of 369.9μM ( Figure 2 (C)). The inhibitory effect is relatively weak. From the above results, it can be seen that AN2718, as an inhibitor of KPC-2 enzyme, shows a strong inhibitory effect at a lower concentration, and the effect is significant. At the same time, it also has different degrees of inhibitory effects on new KPC mutants discovered in recent years, such as KPC-41 and KPC-93, showing a wide range of inhibitory effects. In addition, the chemical structure of AN2718 is simple and easy to modify and synthesize. It is expected that more potent inhibitors against KPC enzymes will be developed, providing possible solutions for future clinical responses to bacterial resistance.

[0075] Molecular docking results

[0076] The docking results of drug AN2718 and KPC-2 enzyme are as follows Figure 3 As shown in Figure 2, the binding energy between AN2718 and KPC-2 protein is -4.344 kcal / mol. Figure 3 (A) It can be seen that AN2718 forms hydrogen bonds with serine 70 (SER70) and serine 130 (SER130) of KPC-2, with bond lengths of 2.3 and In addition, there are polar interactions between serine at position 70 (SER70), serine at position 130 (SER130), asparagine at position 132 (ASN132), asparagine at position 170 (ASN170), threonine at position 237 (THR237) and AN2718; cysteine ​​at position 69 (CYS69), threonine at position 105 (TRP105), leucine at position 167 (LEU167), cysteine ​​at position 238 (CYS238) and other amino acid residues interact with each other through hydrophobic interactions; there are electrostatic interactions between lysine at position 73 (LYS73) and glutamic acid at position 166 (GLU166) and AN2718. The hydrogen bonds and electrostatic interactions formed between AN2718 and the amino acid residues at the active site of the KPC-2 enzyme can significantly increase the affinity between AN2718 and the KPC-2 enzyme. At the same time, contact with the hydrophobic amino acid residues in the active site will release water molecules in the hydrophobic region, leading to an increase in system entropy, thereby reducing the change in free energy and increasing the affinity of the ligand. Figure 3 (B) It can be seen that AN2718 is tightly bound to the KPC-2 enzyme and is located inside the active cavity. The shapes of the substrate structure and the enzyme active cavity structure complement each other, which is consistent with the induced fit theory. Through molecular docking, we theoretically proved that AN2718 can interact with the KPC-2 enzyme and found that the binding between the two is stabilized by multiple forces such as hydrogen bonds, hydrophobic interactions, and electrostatic forces.

[0077] Animal testing results

[0078] The experimental results of AN2718 and meropenem treatment of mice are as follows Figure 4As shown. 24 hours after Hvkp2 infection, all mice in the control group died, with a survival rate of 0%; 3 mice died in the 16mg / kg AN2718 group, with a survival rate of 40%; 4 mice died in the 16mg / kg meropenem group, with a survival rate of only 20%. From 36h to 132h, the survival rate of mice in the AN2718 group was significantly higher than that in the meropenem group. When 16mg / kg AN2718 and 8mg / kg meropenem were used in combination, three mice survived, with a survival rate of 60%. When 8mg / kg AN2718 and 16mg / kg meropenem were used in combination, three mice survived, with a survival rate of 60%. However, it can be seen from the figure that the survival rate of the high-dose AN2718 group was higher than that of the low-dose group in these two groups from 36h to 60h. When 16mg / kg AN2718 and 16mg / kg meropenem were used in combination, 4 mice survived, with a survival rate of 80%. Compared with the control group, both AN2718 and meropenem alone can reduce the mortality of mice to varying degrees. The combination of the two can further reduce the mortality of mice. The survival rate of the high-dose AN2718 and high-dose meropenem combination group was 60% higher than that of the meropenem monotherapy group, 40% higher than that of the AN2718 monotherapy group, and 20% higher than the high-dose and low-dose combinations of the two. The log-rank test (Mantel Cox) test and the log-rank test of the trend test showed that the p values ​​were 0.0107 and 0.0023, respectively, indicating that the effect of the treatment group was significantly different from that of the control group, proving that AN2718 combined with meropenem has an excellent therapeutic effect on mice infected with Hvkp2, further indicating that AN2718 as a KPC-2 inhibitor not only has an excellent inhibitory effect in vitro, but also can play its role in reversing drug resistance in vivo, restoring the bactericidal effect of meropenem, and is a highly potential KPC-2 inhibitor with broad clinical application prospects.

Claims

1. A method for non-diagnostic and non-therapeutic purposes of at least partially inhibiting the function of Klebsiella pneumoniae carbapenemase-2 (KPC-2) or a variant thereof, the method comprising contacting the KPC-2 with a benzoxaborolane selected from the group consisting of 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane, 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane and mixtures thereof. 2 . The method according to claim 1 , wherein the benzoxaborolane is 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane.

3. The method of claim 1, wherein the variant of KPC-2 is KPC-93.

4. The method of claim 1, wherein the benzoxaborolane is 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane and the variant of KPC-2 is KPC-93.

5. Use of a β-lactam antibiotic and a benzoxaborolane in the preparation of a medicament for treating a bacterial infection in a subject in need thereof, wherein the benzoxaborolane is selected from the group consisting of 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane, 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane and mixtures thereof, wherein the bacterial infection is caused by a bacterium expressing Klebsiella pneumoniae carbapenemase-2 (KPC-2) or a variant thereof.

6. The use according to claim 5, wherein the bacterium is Aeromonas caviae, Aeromonas hydrophila, Aeromonas veroni, Citrobacter amalonaticus, Citrobacter freundii, Citrobacter koseri, Citrobacter portucalensis, Citrobacter youngae, Enterobacter asburiae, Enterobacter cloacae, Enterobacter hormaechei, Enterobacter kobei, Escherichia coli, Klebsiella aerogenes, Klebsiella michiganensis, michiganensis, Klebsiella oxytoca, Klebsiella pneumoniae, Klebsiella quasipneumoniae, Morganella morganii, Proteus mirabilis, Pseudomonas aeruginosa, Raoultella planticola, Salmonella enterica, or Serratia marcescens.

7. The use according to claim 5, wherein the bacterium is Escherichia coli, Enterobacter cloacae, Klebsiella aerogenes, carbapenem-resistant Klebsiella pneumoniae or highly virulent Klebsiella pneumoniae.

8. The use according to claim 5, wherein the β-lactam antibiotic is a cephalosporin, a carbapenem, a penicillam or a monobactam.

9. The use according to claim 5, wherein the β-lactam antibiotic is a carbapenem.

10. The use according to claim 5, wherein the β-lactam antibiotic is a carbapenem selected from the group consisting of biapenem, ertapenem, doripenem, imipenem, meropenem, tebipenem and panipenem, or a cephamycin, such as cefoxitin, cefmetazole and cefotetan.

11. The use according to claim 5, wherein the carbapenem is meropenem.

12. The use according to claim 5, wherein the benzoxaborolane is 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane.

13. The use according to claim 5, wherein the variant of KPC-2 is KPC-93.

14. The use according to claim 5, wherein the bacteria is carbapenem-resistant Klebsiella pneumoniae, highly virulent Klebsiella pneumoniae, Escherichia coli or Enterobacter cloacae, the benzoxaborol is 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborol, and the β-lactam antibiotic is a carbapenem selected from the group consisting of biapenem, ertapenem, doripenem, imipenem, meropenem, tebipenem and panipenem, or a cephamycin, such as cefoxitin, cefmetazole and cefotetan.

15. The use according to claim 5, wherein the bacteria is carbapenem-resistant Klebsiella pneumoniae or highly virulent Klebsiella pneumoniae, the benzoxaborol is 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborol, and the β-lactam antibiotic is a carbapenem, which is meropenem.

16. The use according to claim 15, wherein the variant of KPC-2 is KPC-93.

17. Use of a benzoxaborolane selected from the group consisting of 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane, 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane and mixtures thereof in the preparation of a medicament for at least partially inhibiting the function of Klebsiella pneumoniae carbapenemase-2 (KPC-2) or a variant thereof.

18. The use according to claim 17, wherein the benzoxaborolane is 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane.

19. The use according to claim 17, wherein the variant of KPC-2 is KPC-93.

20. The use according to claim 17, wherein the benzoxaborolane is 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborolane and the variant of KPC-2 is KPC-93.

Images