Penicillin G hydroxamic acid derivative and application thereof in prevention and treatment of bacterial infection

Abstract

The invention relates to penicillin G hydroxamic acid, application of the penicillin G hydroxamic acid serving as a metal beta-lactamase inhibitor in preparation of a medicine for reversing bacterial drug resistance, and the penicillin G hydroxamic acid can also be combined with beta-lactam antibiotics for use, so that the penicillin G hydroxamic acid has relatively good synergistic antibacterial activity on drug-resistant strains for expressing MBLs. The penicillin G hydroxamic acid can be used as a metal beta-lactamase inhibitor to be combined with beta-lactam antibiotics for preparing drugs for resisting drug-resistant bacteria and preventing and treating bacterial infection. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and relates to penicillin G hydroxamic acid derivatives and their application in preventing and treating bacterial infections. Background Art

[0002] β-lactam antibiotics refer to a large class of antibiotics with a β-lactam ring in their chemical structure, including the most commonly used penicillins, cephalosporins, and carbapenems in clinical practice, as well as newly developed cephamycins, thienamycins, monocyclic β-lactam antibiotics and other atypical β-lactam antibiotics. Such antibiotics have the advantages of strong bactericidal activity, low toxicity, wide indications, and good clinical efficacy. The mechanisms of action of various β-lactam antibiotics are similar. They can all inhibit the cell wall mucopeptide synthetase, that is, penicillin binding proteins (PBPs), thereby hindering the synthesis of cell wall mucopeptides, causing bacterial cell wall defects, and bacterial cells to expand and lyse. In addition, the lethal effect on bacteria should also include triggering the autolytic enzyme activity of bacteria, and mutant strains lacking autolytic enzymes show drug resistance.

[0003] Metallo-β-lactamases (MBLs) are the key enzymes for bacteria to be resistant to β-lactam antibiotics. The active site contains one or two metal Zn ions. According to the similarity of gene coding sequences, they can be divided into classes B1, B2, and B3. Among them, class B1 is the most common and widely disseminated MBLs in clinical practice, including types such as NDM, IMP, and VIM. The active site contains 2 metal Zn ions, which can hydrolyze almost all β-lactam antibiotics. Currently, it is becoming more and more common in clinical practice, and clinical treatment is extremely difficult. Metallo-β-lactamase inhibitors (MBLIs) can inhibit the activity of MBLs, thereby restoring the effectiveness of β-lactam antibiotics against bacteria resistant to MBLs. So far, no MBLIs have been marketed for clinical use, which is an unmet clinical need. Therefore, the research and development of MBLIs is an important breakthrough point for the research and development of new antibacterial drugs, with very good clinical application prospects and social and economic benefits.

[0004] There have been many reports on MBLIs, but most of the reported MBLIs have the following problems, leading to a dim prospect for drug development. First, since some of the human own metalloproteins are members of the MBLs superfamily, overly broad-spectrum and non-selective MBLIs such as metal chelators EDTA and mercaptoacetic acid can act on these proteins and cause side effects. Second, some MBLIs have a strong inhibitory effect on enzymes in enzyme inhibition experiments, but the effect is not ideal when combined with β-lactam antibiotics against Gram-negative bacteria expressing MBLs. Therefore, it is crucial to find new MBLIs with obvious curative effects, safety and no toxicity.

[0005] The mechanisms of hydrolysis of various β-lactam antibiotics by metallo-β-lactamases are similar, that is: the zinc ion in the active center of the metallo-β-lactamase coordinates with the carbonyl oxygen atom of the β-lactam ring, changing the electron cloud density of the β-lactam ring, thereby weakening the stability of the carbon-nitrogen bond in the β-lactam ring; at the same time, water molecules are polarized under the action of metal ions to form nucleophilic hydroxyl groups, and the nucleophilic hydroxyl groups attack the carbonyl carbon atom of the β-lactam ring, undergoing a nucleophilic substitution reaction, resulting in the opening of the β-lactam ring, and further causing the β-lactam antibiotics to lose their antibacterial activity. Summary of the Invention

[0006] Based on this, the object of the present invention is penicillin G hydroxamic acid and its application in preventing and treating bacterial infections. The penicillin G hydroxamic acid is used as a metallo-β-lactamase inhibitor and can be combined with β-lactam antibiotics to prevent and treat infections of drug-resistant bacteria.

[0007] The first aspect of the present invention is to provide penicillin G hydroxamic acid, which has the following chemical structural formula:

[0008]

[0009] The second aspect of the present invention is to provide the application of penicillin G hydroxamic acid as a metallo-β-lactamase inhibitor in the preparation of drugs for reversing bacterial drug resistance, and the bacteria are drug-resistant bacteria expressing metallo-β-lactamases (MBLs).

[0010] In some of these embodiments, the metallo-β-lactamase is NDM-1 or VIM-2 type.

[0011] The third aspect of the present invention is to provide the application of penicillin G hydroxamic acid as a metallo-β-lactamase inhibitor in the preparation of drugs for reversing bacterial drug resistance, or the application of penicillin G hydroxamic acid as defined in claim 1 as a metallo-β-lactamase inhibitor combined with β-lactam antibiotics in the preparation of drugs against drug-resistant bacteria, and the bacteria are drug-resistant bacteria expressing metallo-β-lactamases.

[0012] In some of these embodiments, the drug-resistant bacteria expressing metallo-β-lactamase are drug-resistant Gram-negative bacteria expressing metallo-β-lactamase; preferably, the drug-resistant bacteria expressing metallo-β-lactamase are drug-resistant Gram-negative bacilli expressing metallo-β-lactamase; more preferably, the drug-resistant bacteria expressing metallo-β-lactamase are drug-resistant Escherichia coli expressing metallo-β-lactamase.

[0013] In some of these embodiments, the β-lactam antibiotic is a carbapenem antibiotic.

[0014] In some of these embodiments, the carbapenem antibiotic is meropenem.

[0015] A fourth aspect of the present invention is to provide a combined drug for preventing and treating drug-resistant bacteria, the active ingredients of which include the penicillin G hydroxamic acid and the β-lactam antibiotic, the penicillin G hydroxamic acid and the β-lactam antibiotic are respectively independent dosing units, or the penicillin G hydroxamic acid and the β-lactam antibiotic jointly form a combined dosing unit, and the bacteria are drug-resistant bacteria expressing metallo-β-lactamase.

[0016] The present invention has synthesized a new compound, penicillin G hydroxamic acid, and found that this penicillin G hydroxamic acid has good inhibitory activity (IC50 value) against MBLs (NDM-1, VIM-2), and can be used as an application of a metallo-β-lactamase inhibitor in the preparation of antibacterial drugs. The penicillin G hydroxamic acid can also be used in combination with a carbapenem antibiotic (such as meropenem) in the β-lactam antibiotics to have good synergistic antibacterial activity (FICI value) against drug-resistant strains expressing MBLs (NDM-1, VIM-2). The penicillin G hydroxamic acid can be used as a metallo-β-lactamase inhibitor in combination with a β-lactam antibiotic for the preparation of drugs against drug-resistant bacteria to prevent and treat bacterial infections. Description of the Drawings

[0017] Figure 1 is the high performance liquid chromatography (HPLC) spectrum of penicillin G hydroxamic acid in Example 1.

[0018] Figure 2 is the mass spectrum of penicillin G hydroxamic acid in Example 1.

[0019] Figure 3 The hydrogen spectrum of penicillin G hydroxamic acid in Example 1.

[0020] Figure 4 Time-kill curves of penicillin G hydroxamic acid and meropenem alone or in combination against the constructed strain of E. coli (NDM-1), where W-1: penicillin G hydroxamic acid; MEM: meropenem.

[0021] Figure 5 The cytotoxic effect of penicillin G isohydroxamic acid on HEK-293T cells, and the P value was determined by unpaired t-test, **P < 0.01 vs. control.

[0022] Figure 6 The cytotoxic effect of penicillin G isohydroxamic acid on L-O2 cells, and the P value was determined by unpaired t-test, *P < 0.05 vs. control.

[0023] Figure 7 Hemolytic toxicity of penicillin G isohydroxamic acid at different concentrations. Detailed implementation manners

[0024] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0025] For the experimental methods without specific conditions noted in the following examples, they are usually carried out under conventional conditions. For example, the fourth edition of "Molecular Cloning: A Laboratory Manual" edited by Green and Sambrook was published in 2013, or according to the conditions recommended by the manufacturer. All kinds of common chemical reagents used in the examples are commercially available products.

[0026] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0027] Definitions For the convenience of understanding the present technology, some terms and phrases are defined below.

[0028] Escherichia coli (E. coli), Escherichia coli, belongs to Gram-negative short bacilli.

[0029] MEM: Meropenem, with the chemical formula C 17 H 25 N 3 O 5 S, a broad-spectrum carbapenem antibiotic, is a β-lactam antibiotic.

[0030] In some embodiments of the present invention, it involves the design and synthesis of penicillin G isohydroxamic acid.

[0031] In some embodiments of the present invention, it relates to the study of the in vitro MBLs inhibitory activity of penicillin G isohydroxamic acid, including

[0032] 1) Construction of recombinant engineering bacteria

[0033] 2) Isolation and purification of MBLs

[0034] 3) Determination of the IC50 of the enzyme inhibitory activity of penicillin G isohydroxamic acid.

[0035] In some embodiments of the present invention, it relates to the antibacterial experiment of the combination of penicillin G isohydroxamic acid and β-lactam antibiotics: the MIC (minimum inhibitory concentration) value of meropenem alone or in combination with penicillin G isohydroxamic acid against MBLs-producing Escherichia coli was determined by the microbroth dilution method. Meropenem was diluted to 128 - 0.25 μg / mL, and the concentration of penicillin G isohydroxamic acid was 128 - 2 μg / mL. At the same time, the combination of meropenem and captopril (classical MBLI) was set as a positive control. Incubate at 37 °C for 16 - 20 h and record the MIC value.

[0036] In some embodiments of the present invention, it relates to the cytotoxicity experiment and hemolysis experiment of penicillin G isohydroxamic acid, including: The cytotoxic effects of penicillin G isohydroxamic acid at different concentrations (128 - 4 μg / mL) on human embryonic kidney cells 293T (HEK - 293T) and human normal liver cells (L - O2) were determined by the CCK - 8 method, and the hemolysis rate of penicillin G isohydroxamic acid at seven different concentrations (128 - 4 μg / mL) was determined. For the cytotoxicity experiment, cells untreated with the compound were set as a positive control group, and for the hemolysis experiment, red blood cells treated with 0.5% Triton X - 100 were set as a positive control group.

[0037] The content of the present invention will be further described in detail through specific embodiments below.

[0038] Example 1: Design and synthesis of penicillin G isohydroxamic acid

[0039] Penicillin G isohydroxamic acid derivatives can be obtained by condensing penicillin G with protected hydroxylamine and then deprotecting.

[0040] Step 1: Dissolve potassium penicillin G (1.0 g, 2.87 mM) in 5 mL of DMF (N,N - dimethylformamide), add 1 - (3 - dimethylaminopropyl) - 3 - ethylcarbodiimide hydrochloride (EDCI) (822 mg, 4.3 mM), 1 - hydroxybenzotriazole (HOBT) (580 mg, 4.3 mM) and O - (tetrahydro - 2H - pyran - 2 - yl)hydroxylamine (THP - O - NH 2, 1.3 g, 5.73 mM), and the reaction was carried out for 1 h. LC-MS showed that the reaction was completed. The crude product was purified by preparative liquid chromatography to obtain 200 mg (crude) of a colorless oil.

[0041] Step 2: Pyridinium p-toluenesulfonate (PPTS, 450 mg, 0.82 mM) was dissolved in anhydrous ethanol (3 mL). Then the mixture was stirred at 50 °C for 16 h. After purification of the crude product by preparative high-performance liquid chromatography, a white solid product (12 mg) was obtained.

[0042] Synthetic route:

[0043]

[0044] The high-performance liquid chromatography spectrum, mass spectrum, and hydrogen spectrum of penicillin G hydroxamic acid are shown in Figure 1 and Figure 2 and Figure 3 .

[0045] Penicillin G hydroxamic acid (white powder, purity 98.78%, M.W. 349.41), 1H NMR (400 MHz, DMSO-d6) δ 10.68 (s, 1H), 7.35 - 7.23 (m, 5H), 5.75 (s, 1H), 4.51 (s, 1H), 4.28 (s, 1H), 3.70 (d, J = 14.8 Hz, 1H), 3.57 (d, J = 16.4 Hz, 1H), 1.38 - 1.32 (m, 1H).

[0046] The prepared penicillin G hydroxamic acid was used in the following experiments.

[0047] Example 2: Study on the in vitro inhibitory activity of penicillin G hydroxamic acid against MBLs

[0048] (1) Construction of recombinant engineering bacteria

[0049] Recombinant plasmids (NDM-1, VIM-2, and IMP-1 recombinant plasmids) and E. coli BL21(DE3) were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0050] Take out the BL21(DE3) competent cells from the -80°C ultra-low temperature refrigerator and let them thaw on ice. In a laminar flow hood, take 1 μL of the recombinant plasmid (NDM-1, VIM-2, and IMP-1 recombinant plasmids) and add it to 100 μL of BL21(DE3) competent cells, and gently pipette to mix evenly. Then incubate the competent cells on ice for 30 min, heat shock at 42°C for 90 s, then incubate on ice for 2 min. Then add 900 μL of LB liquid medium to the competent cells and culture them in a constant temperature shaking incubator at 37°C and 220 rpm for 1 h. Spread the competent cell suspension evenly on an LB solid medium containing 25 μg / mL kanamycin and incubate it upside down at 37°C overnight.

[0051] (2) Isolation and purification of MBLs

[0052] 1) Preparation of solutions

[0053] Lysis buffer: Weigh 5.95 g of HEPES and dissolve it in 500 mL of sterile distilled water to make the final concentration of the buffer 50 mM. Subsequently, add NaCl with a final concentration of 300 mM and 0.1% Triton X-100 to the buffer, and adjust the pH to about 8.0 with 1 M NaOH solution.

[0054] Binding buffer: Add NaCl with a final concentration of 300 mM and 0.2% Triton X-100 to a 50 mM HEPES buffer and adjust the pH to about 8.0.

[0055] Washing buffer: Add NaCl with a final concentration of 300 mM, 50 mM imidazole, and 100 μM ZnCl2 to a 50 mM HEPES buffer and adjust the pH to about 8.0.

[0056] Elution buffer: Add NaCl with a final concentration of 300 mM, 250 mM imidazole, and 100 μM ZnCl2 to a 50 mM HEPES buffer and adjust the pH to about 8.0.

[0057] Exchange buffer 1: Weigh 2.98 g of HEPES and dissolve it in 500 mL of sterile distilled water to make the final concentration of the buffer 25 mM, and adjust the pH to about 7.0. And add NaCl with a final concentration of 300 mM and 100 μM ZnCl 2 .

[0058] Displacement buffer 2: Add NaCl with a final concentration of 200 mM and ZnCl with a final concentration of 100 μM to 25 mM HEPES buffer, and adjust the pH to about 7.0. 2

[0059] Displacement buffer 3: Add NaCl with a final concentration of 100 mM and ZnCl with a final concentration of 100 μM to 25 mM HEPES buffer, and adjust the pH to about 7.0. 2

[0060] 2) Expression and purification of three clinically common metallo-β-lactamases (NDM-1, VIM-2, IMP-1)

[0061] ① Expression and purification of NDM-1

[0062] Resuscitation: Use an inoculation loop to pick up a loop of glycerol bacteria carrying the NDM-1 gene plasmid, inoculate it onto an LB agar plate containing 25 μg / mL KAN, and incubate it overnight at 37°C in a constant temperature shaking incubator for 12 - 16 h.

[0063] Activation: Use an inoculation loop to pick a single colony of Escherichia coli expressing NDM-1 into 10 mL of LB liquid medium containing 25 μg / mL kanamycin, and incubate it overnight at 37°C and 150 rpm on a shaker for 12 - 16 h.

[0064] Scale-up culture: Take 2.5 mL of the bacterial liquid from the previous step and add it to 250 mL of LB liquid medium containing 25 μg / mL kanamycin (two portions), and culture it at 37°C and 150 rpm on a shaker for about 4 h until the OD value of the bacterial liquid concentration is 0.6 - 0.8.

[0065] Low-temperature induction culture: Add 250 μL of 0.1 M IPTG to the bacterial liquid prepared in the previous step, and perform low-temperature induction culture at 18°C and 150 rpm for about 20 h.

[0066] Bacterial cell collection: Centrifuge the bacterial liquid after low-temperature induction at 10,000 rpm, 4°C for 5 min. Discard the supernatant culture medium, resuspend the bacterial cell precipitate with an appropriate amount of sterile PBS and then centrifuge (10,000 rpm, 4°C, 5 min), remove the supernatant PBS, and store the bacterial cell precipitate at -80°C.

[0067] Bacterial cell disruption: Resuspend the frozen bacterial cell precipitate from the previous step with an appropriate amount of lysis buffer, and add the corresponding volume of protease inhibitor (x100). Subsequently, place the resuspended bacterial liquid in an ice bath and use a cell ultrasonic disruptor for ultrasonic treatment. Under the condition of 400 W, ultrasonic for 5 s and interval for 5 s, with a total ultrasonic time of 15 min.

[0068] Centrifugation: The broken bacterial solution was centrifuged at 12,000 rpm, 4 °C for 3 min. The supernatant was collected and filtered through a 0.45 μM microporous filter membrane to obtain the crude protein. In addition, a small amount of bacterial precipitate was collected for subsequent detection.

[0069] Nickel column affinity purification: First, the nickel column was washed and equilibrated with 5 mL of Binding buffer, and then the crude protein sample collected in the previous step was loaded onto the nickel column and circulated once. Subsequently, the column was washed with Washing buffer containing 50 mM imidazole to remove the miscellaneous proteins, with a total of 6 washes; then eluted with Elution buffer containing 250 mM imidazole, and the eluate was collected. Finally, the nickel column was treated and stored in 20% ethanol.

[0070] SDS-PAGE gel electrophoresis: Samples such as the crude protein and eluate collected in the previous step were subjected to SDS-PAGE gel electrophoresis, stained with Coomassie Brilliant Blue Fast Stain, and the protein purification was identified and collected.

[0071] Dialysis: The purified protein was placed in a dialysis bag. First, it was dialyzed with replacement buffer 1 for 2 - 4 h, then replaced with replacement buffer 2 and continued dialysis. After 6 - 8 h, it was replaced with replacement buffer 3 and dialyzed for 12 h. After 12 h of dialysis, it was replaced with replacement buffer 3 again and continued dialysis for 2 h. The dialyzed protein sample was collected and the target protein was collected by filtering through a 0.22 μm microporous filter membrane. The collected target protein was subjected to subsequent enzyme activity detection, aliquoted and stored at -20 °C.

[0072] ② Expression and purification of VIM-2

[0073] Resuscitation: A loop of glycerol bacteria carrying the VIM-2 gene plasmid was taken with an inoculation loop and inoculated onto an LB agar plate containing 25 μg / mL kanamycin, and then inverted and incubated in a 37 °C constant temperature shaking incubator overnight for 12 - 16 h.

[0074] Activation: A single colony of Escherichia coli expressing VIM-2 was picked with an inoculation loop into 10 mL of LB liquid medium containing 25 μg / mL kanamycin, and incubated at 37 °C, 150 rpm on a shaker overnight for 12 - 16 h.

[0075] Scale-up culture: 2.5 mL of the bacterial solution from the previous step was added to 250 mL of LB liquid medium containing 25 μg / mL kanamycin (two portions), and cultured at 37 °C, 150 rpm on a shaker for about 4 h until the OD value of the bacterial solution concentration was 0.6 - 0.8.

[0076] Low-temperature induction culture: 250 μL of 0.1 M IPTG was added to the bacterial solution prepared in the previous step, and cultured at 18 °C, 150 rpm for about 20 h under low-temperature induction.

[0077] Bacterial cell collection: Centrifuge the bacterial liquid after low-temperature induction at 10,000 rpm, 4°C for 5 min. Discard the supernatant medium. Resuspend the bacterial cell pellet with an appropriate amount of sterile PBS and then centrifuge (10,000 rpm, 4°C, 5 min). Remove the supernatant PBS, and store the bacterial cell pellet at -80°C.

[0078] Bacterial cell disruption: Resuspend the frozen bacterial cell pellet from the previous step with an appropriate amount of lysis buffer and add the corresponding volume of protease inhibitor (x100). Subsequently, place the resuspended bacterial liquid in an ice bath and perform ultrasonic treatment using a cell ultrasonic disruptor. Under the condition of 400 W, ultrasonicate for 5 s with a 5-s interval, and the total ultrasonication time is 15 min.

[0079] Centrifugation: Centrifuge the disrupted bacterial liquid at 12,000 rpm, 4°C for 3 min. Collect the supernatant. Filter the supernatant through a 0.45 μM microporous filter membrane to obtain the crude protein. Additionally, collect a small amount of bacterial cell pellet for subsequent detection.

[0080] Nickel column affinity purification: First, wash and equilibrate the nickel column with 5 mL of Binding buffer, then load the crude protein sample collected in the previous step onto the nickel column and repeat the flow-through once. Subsequently, wash the column with Washing buffer containing 50 mM imidazole to wash away the impurity proteins, with a total of 6 washes; then elute with Elution buffer containing 250 mM imidazole, collect the eluate, and finally treat the nickel column and store it in 20% ethanol.

[0081] SDS-PAGE gel electrophoresis: Perform SDS-PAGE gel electrophoresis on samples such as the crude protein and eluate collected in the previous step, stain with Coomassie Brilliant Blue rapid staining solution, identify the protein purification situation and collect.

[0082] Dialysis: Place the purified protein in a dialysis bag. First, dialyze with replacement buffer 1 for 2 - 4 h, then change to replacement buffer 2 and continue dialysis. After 6 - 8 h, change to replacement buffer 3 and continue dialysis for 12 h. After 12 h of dialysis, change to replacement buffer 3 again and continue dialysis for 2 h. Collect the dialyzed protein sample and filter through a 0.22 μm microporous filter membrane to collect the target protein. Perform subsequent enzyme activity detection on the collected target protein, aliquot it, and store it at -20°C.

[0083] ③ Expression and purification of IMP-1

[0084] Resuscitation: Use an inoculation loop to pick up a loop of glycerol bacteria carrying the IMP-1 gene plasmid and inoculate it onto an LB agar plate containing 25 μg / mL kanamycin. Invert and incubate it overnight at 37°C in a constant temperature shaking incubator for 12 - 16 h.

[0085] Activation: Pick a single colony of Escherichia coli expressing IMP-1 with an inoculation loop and transfer it to 10 mL of LB liquid medium containing 25 μg / mL kanamycin. Incubate overnight at 37°C and 150 rpm on a shaker for 12 - 16 h.

[0086] Enlarged culture: Take 2.5 mL of the bacterial solution from the previous step and add it to two 250 mL portions of LB liquid medium containing 25 μg / mL kanamycin. Culture at 37°C and 150 rpm on a shaker for about 4 h until the OD value of the bacterial solution concentration reaches 0.6 - 0.8.

[0087] Low-temperature induction culture: Add 250 μL of 0.1 M IPTG to the bacterial solution prepared in the previous step and perform low-temperature induction culture at 18°C and 150 rpm for about 20 h.

[0088] Bacterial cell collection: Centrifuge the bacterium solution after low-temperature induction at 10,000 rpm, 4°C for 5 min. Discard the supernatant culture medium. Resuspend the bacterial cell precipitate with an appropriate amount of sterile PBS and then centrifuge (10,000 rpm, 4°C, 5 min). Remove the supernatant PBS, and store the bacterial cell precipitate at -80°C.

[0089] Disrupt bacterial cells: Resuspend the frozen bacterial cell precipitate from the previous step with an appropriate amount of lysis buffer and add the corresponding volume of protease inhibitor (x100). Subsequently, place the resuspended bacterium solution in an ice bath and use a cell ultrasonic disruptor for ultrasonic treatment. Under the condition of 400 W, ultrasonic for 5 s and interval for 5 s, with a total ultrasonic time of 15 min.

[0090] Centrifugation: Centrifuge the disrupted bacterium solution at 12,000 rpm, 4°C for 3 min. Collect the supernatant. Filter the supernatant through a 0.45 μM microporous filter membrane to obtain the crude protein. Additionally, collect a small amount of bacterial precipitate for subsequent detection.

[0091] Nickel column affinity purification: First, wash and balance the nickel column with 5 mL of Binding buffer, then load the crude protein sample collected in the previous step onto the nickel column and circulate it once. Subsequently, wash the column with Washing buffer containing 50 mM imidazole to wash away the impurity proteins, with a total of 6 washes; then elute with Elution buffer containing 250 mM imidazole, collect the eluate, and finally treat the nickel column and store it in 20% ethanol.

[0092] SDS-PAGE gel electrophoresis: Perform SDS-PAGE gel electrophoresis on the samples such as the crude protein and eluate collected in the previous step, stain with Coomassie Brilliant Blue rapid staining solution, identify the protein purification situation and collect the protein.

[0093] Dialysis: The purified protein was placed in a dialysis bag. First, it was dialyzed with replacement buffer 1 for 2 - 4 h, then replaced with replacement buffer 2 and continued to be dialyzed. After 6 - 8 h, it was replaced with replacement buffer 3 and dialyzed for 12 h. After 12 h of dialysis, it was replaced with replacement buffer 3 again and dialyzed for 2 h. The protein sample after dialysis was collected, and the target protein was collected by filtration through a 0.22 μm microporous filter membrane. The collected target protein was subjected to subsequent enzyme activity detection, aliquoted, and stored at -20 °C.

[0094] (3) Enzyme inhibition activity IC 50 Determination

[0095] 1) Enzyme kinetics experiment

[0096] ① Experimental method

[0097] The enzyme kinetics experiment was carried out in a 96-well plate. The hydrolysis substrate was selected as nitrocefin, and the detection wavelength was 492 nm. The final concentration of nitrocefin was 1 - 30 μM, and then the MBLs solution (the final concentrations of NDM-1, VIM-2, and IMP-1 were 10 nM) was added. After mixing, the change in absorbance was immediately detected. It was detected once every 10 s for a total of 3 min. Each group of experiments was determined in parallel at least three times. The hydrolysis rate in the first 30 s before the reaction was used for the calculation of Km.

[0098] ② Experimental results

[0099] The Km values of the three MBLs (NDM-1, VIM-2, and IMP-1) are shown in Table 1.

[0100] Table 1 Km values of the three MBLs

[0101]

[0102] 2) IC 50 Determination

[0103] ① Experimental method

[0104] Penicillin G hydroxamic acid was dissolved in sterile distilled water and then diluted with the corresponding buffer. The concentration range of penicillin G hydroxamic acid obtained in Example 1 was 0 - 300 μM, and the concentration of the substrate nitrocefin was 10 μM. Different concentrations of penicillin G hydroxamic acid were pre-incubated with the enzyme for 15 min first, then the substrate was added to initiate the reaction. After pipetting and mixing, the change in absorbance was immediately measured at 492 nm. It was measured once every 10 s for 1 min. The increase in the amount of hydrolysis product in the first 30 s was used for the calculation of the initial reaction rate. Each group of experiments was determined in parallel at least three times.

[0105] Calculate the residual activity of MBLs after adding different concentrations of inhibitors. The residual activity R (100%) of the enzyme = (V i / V0 ) x 100% (V i : Rate of enzymatic hydrolysis of the substrate in the presence of the inhibitor; V 0 : Rate of enzymatic hydrolysis of the substrate without the inhibitor). And a non-linear fitting graph of inhibitor concentration vs. residual activity of the enzyme was plotted using Graphpad Prism 8.0 to obtain the IC 50 value.

[0106] ② Experimental results

[0107] The IC 50 determination results of penicillin G hydroxamic acid against three MBLs (NDM-1, VIM-2, and IMP-1) are shown in Table 2: Penicillin G hydroxamic acid showed good inhibitory activity against NDM-1 and VIM-2, with IC 50 values of 70.28 μM and 76.59 μM, respectively. However, for IMP-1, penicillin G hydroxamic acid did not show inhibitory activity.

[0108] Table 2 IC 50 and Ki of penicillin G hydroxamic acid against three MBLs

[0109]

[0110] Example 3: Antibacterial experiment of penicillin G hydroxamic acid combined with β-lactam antibiotics

[0111] 1) Checkerboard experiment:

[0112] ① Experimental method

[0113] The experimental strains were constructed strains:

[0114] E. coli BL21(DE3) / pET26b-NDM-1 is abbreviated as E. coli (NDM-1)

[0115] E. coli BL21(DE3) / pET26b-VIM-2 is abbreviated as E. coli (VIM-2)

[0116] E. coli BL21(DE3) / pET26b-IMP-1 is abbreviated as E. coli (IMP-1)

[0117] The MIC values of meropenem / inhibitor (penicillin G hydroxamic acid or captopril or DMSO) alone and in combination against three constructed strains were determined by the microbroth dilution method established by the Clinical and Laboratory Standards Institute (CLSI) of the United States. The combined antibacterial experiment was achieved through a checkerboard experiment, that is, by preparing dilution plates for the combined use of two drugs: A stock solution of meropenem (MEM) at 5,120 μg / mL and a stock solution of inhibitor were prepared with sterile distilled water and stored at -20 °C for later use. Under sterile conditions, for the gradient dilution of MEM in the horizontal direction of a 96-well plate, 100 μL of MEM or inhibitor solution (512 μg / mL) was added to the first column of the 96-well plate, 50 μL of culture medium was added to the 2nd - 11th columns. 50 μL of the solution was aspirated from the first column and transferred to the second column, and mixed by pipetting. In the same way, 50 μL of the mixed solution was aspirated from the second column and transferred to the third column and mixed again. Such continuous operation was carried out until the 11th column (0.5 - 512 μg / mL), and finally 50 μL of the mixed solution was aspirated and discarded. In the vertical direction, 50 μL of the inhibitor diluted to different concentrations (512 - 8 μg / mL) with the culture medium in advance was added. The culture solution concentration of the above bacteria was adjusted to 0.5 McFarland concentration and then diluted 100 times, and IPTG with a final concentration of 0.5 mM was added. 100 or 150 μL of the bacterial solution was aspirated and added to the 96-well plate so that the final concentration of the bacterial solution was about 5x10 5 CFU / mL, the final concentrations of MEM and the inhibitor were both 0 - 128 μg / mL, and the final volume of each well was 200 μL. The 96-well plate added with the bacterial solution was placed in a constant temperature incubator at 37 °C and cultured overnight for 16 - 20 h, and then the MIC value was recorded. The MIC value was the minimum concentration of the drug where no obvious bacterial growth was observed with the naked eye.

[0118] Calculation of the fractional inhibitory concentration index (FICI): The FICI value can be calculated according to a specific equation, that is, FICI = FIC A + FIC B , FIC A is the MIC value of drug A in combination / the MIC value of drug A alone, FIC B is the MIC value of drug B in combination / the MIC value of drug B alone, which is used to evaluate the interaction between two different drugs when used in combination. If FICI ≤ 0.5, it indicates that the combination of the two drugs has a synergistic effect. If 0.5 < FICI ≤ 4, there is no relevant effect for the combination of the two drugs. If FICI > 4, it indicates that there is an antagonistic effect for the combination of the two drugs. The smaller the FICI index, the stronger the synergistic effect of the drugs.

[0119] ② Experimental results

[0120] The MIC values (shown in Table 3, Table 4, and Table 5 respectively) and FICI values (Table 6) of meropenem alone and in combination with inhibitors against drug-resistant strains expressing NDM-1, VIM-2, and IMP-1 were as follows: The FICI of penicillin G isohydroxamate combined with meropenem against drug-resistant strains expressing NDM-1 and VIM-2 was <0.50, indicating that this combination had good synergistic antibacterial activity against both drug-resistant bacteria, while it had no synergistic antibacterial activity against drug-resistant bacteria expressing IMP-1.

[0121] Table 3 MIC values (μg / mL) of meropenem alone and in combination with inhibitors at different concentrations (μg / mL) against E. coli (NDM-1)

[0122]

[0123] a The MIC of penicillin G isohydroxamate against E. coli (NDM-1) was >1024 μg / mL. b The MIC of captopril against E. coli (NDM-1) was >1024 μg / mL, and the MIC of MEM against E. coli (NDM-1) was 128 μg / mL.

[0124] Table 4 MIC values (μg / mL) of meropenem alone and in combination with inhibitors at different concentrations (μg / mL) against E. coli (VIM-2)

[0125]

[0126] a The MIC of penicillin G isohydroxamate against E. coli (VIM-2) was >1024 μg / mL. b The MIC of captopril against E. coli (VIM-2) was >1024 μg / mL, and the MIC of MEM against E. coli (VIM-2) was 16 μg / mL.

[0127] Table 5 MIC values (μg / mL) of meropenem alone and in combination with inhibitors at different concentrations (μg / mL) against E. coli (IMP-1)

[0128]

[0129] a The MIC of penicillin G isohydroxamate against E. coli (IMP-1) was >1024 μg / mL. b The MIC of captopril against E. coli (IMP-1) was >1024 μg / mL, and the MIC of MEM against E. coli (IMP-1) was 16 μg / mL.

[0130] Table 6 The minimum FICI of the combination of meropenem and penicillin G hydroxamic acid

[0131]

[0132] 2) Time-kill curve

[0133] ① Experimental method

[0134] Using the constructed strain E. coli BL21(DE3) / pET26b-NDM-1 as the experimental strain, set up a meropenem group (32 μg / mL), a penicillin G hydroxamic acid group (64 μg / mL), a combined drug group (32 μg / mL MEM + 64 μg / mL penicillin G hydroxamic acid), and a blank control group (containing only culture medium and bacterial solution). Take four sterile shaking tubes, and add 10 mL of LB liquid medium containing the corresponding drug concentration to each shaking tube. Pipette 100 μL of the bacterial solution with a 0.5 McFarland concentration into 10 mL of LB liquid medium (25 μg / mL kanamycin), and culture it at 37 °C until the logarithmic growth phase (about 4 - 6 h). Subsequently, adjust the concentration of the bacterial solution in the logarithmic growth phase to 1.0 McFarland concentration with LB liquid medium, and add 100 μL of the bacterial solution to each shaking tube. In a 96-well plate, perform 10-fold serial dilutions of each group of bacterial solutions with LB liquid medium. Pipette 10 μL of each dilution and drop it onto an M-H agar plate. After the plate is placed for about 30 min, invert it and incubate it overnight at 37 °C for 16 - 20 h, and observe and record the number of colonies on the plate. Using time as the abscissa and log 10 CFU / mL as the ordinate to plot the curve. The experiments were independently repeated three times.

[0135] ② Experimental results

[0136] The results of the time-kill curve experiment are as Figure 4 shown: In the growth control group, the drug-resistant strains showed a normal upward trend in their bacterial growth curves within 8 hours without the addition of meropenem or penicillin G hydroxamic acid, indicating good growth of the strains; when using 32 μg / mL of meropenem alone, it had a continuous inhibitory effect on the growth of the strains within 2 h, but the number of bacteria gradually increased after 2 h; the effect of 64 μg / mL of penicillin G hydroxamic acid on the drug-resistant bacteria was almost the same as that of the growth control, indicating no inhibitory effect; when 32 μg / mL of meropenem and 64 μg / mL of penicillin G hydroxamic acid were combined and used on this drug-resistant strain, the bacteria decreased by 3 log 10 CFU / mL within 8 h, indicating that penicillin G hydroxamic acid and meropenem have a synergistic bactericidal activity when used in combination against drug-resistant strains expressing NDM-1. See Figure 4 .

[0137] 4. Cytotoxicity Experiment and Hemolysis Experiment of Penicillin G Hydroxamic Acid

[0138] (1) Cytotoxicity Experiment

[0139] 1) Experimental Method

[0140] Observe the morphology and growth status of HEK-293T cells and L-O2 cells under a microscope. After most cells adhere to the wall, perform cell passage. Digest the adherent cells, centrifuge, discard the supernatant, and add HEK-293T cells and L-O 2 Cell-specific medium to 10 mL to obtain a cell suspension. Add 100 μL of the cell suspension to each well of a 96-well plate, and the number of cells per well is approximately 1×10 4 cells. Then place the 96-well plate in a cell culture incubator at 37 °C containing 5% CO 2 and incubate for 24 h. After all cells adhere to the wall, discard the medium and wash each well once with PBS.

[0141] Dissolve penicillin G hydroxamic acid with sterile distilled water, and the concentration of the stock solution is 5,120 μg / mL. Gradient dilute it with cell-specific medium to make the final concentrations of the compound 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, and 4 μg / mL. Then add 100 μL of compounds with different concentrations to the above 96-well plate containing cells, and set 5 replicates for each concentration. At the same time, set a control group and a blank group. The control wells do not contain the compound, and the blank wells do not contain cells. Place the 96-well plate in a constant temperature cell culture incubator at 37 °C containing 5% CO 2 and incubate for 24 h. After the compound and cells are co-incubated in the incubator for 24 h, discard the medium. Dilute the medium and CCK-8 reagent at a ratio of 1:10, calculate the total volume of CCK-8 solution required for the experiment, add 100 μL of CCK-8 solution to each well, and incubate for 1 - 2 h. Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0142] Eliminate outliers caused by bubble interference or other reasons. Calculate the cell survival rate, and the cell survival rate (%) = [(ODexperimental group - ODblank group) / (ODcontrol group - ODblank group)] x 100%.

[0143] Statistical Method: Use Graphpad Prism 8.0 biostatistical software to perform statistical processing on the experimental data. Unpaired t-tests are used for differential analysis. When P < 0.05, it indicates a significant difference (*), and when P < 0.01, it indicates a highly significant difference (**).

[0144] 2) Experimental Results

[0145] The experimental results are asFigure 5 and Figure 6 As shown in Figure 6 , as the concentration of penicillin G hydroxamic acid increased to 128 μg / mL, the survival rate of HEK-293T cells was still greater than 80%. Compared with the positive control group without compound treatment, this difference was statistically significant (P<0.01). For L-O2 cells, the trend of the effect of its activity with the inhibitor concentration was consistent with that of HEK-293T cells, and the difference was also statistically significant (P<0.05), indicating that the compound penicillin G hydroxamic acid was not cytotoxic to these two types of cells.

[0146] (2) Hemolysis experiment:

[0147] 1) Experimental method

[0148] Prepare red blood cell suspension: Take 1 mL of sterile human blood in a centrifuge tube, then resuspend it by pipetting with an equal volume of PBS buffer, centrifuge at 3,000 rpm for 10 min, and discard the supernatant. Dilute the red blood cell pellet 20-fold with PBS buffer to obtain a 5% red blood cell suspension.

[0149] Prepare compound solution: Dissolve penicillin G hydroxamic acid with sterile distilled water, and then dilute it gradiently with PBS buffer. The final concentrations of the compound are 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, and 2 μg / mL respectively. Select 0.5% Triton X-100 as the positive control.

[0150] Add samples: Mark the 96-well plate, add 100 μL of 5% red blood cell suspension to each well, and then add 100 μL of the pre-diluted compound solution with different concentrations to each well. Set 5 parallel replicates. At the same time, set the positive control group and the negative control group.

[0151] Measure the OD value: After the 96-well plate is incubated in a 37°C incubator for 1 hour, take it out, then transfer the solution in each well to a 1.5 mL EP tube respectively, make marks, adjust the centrifuge temperature to 4°C, and centrifuge at 3,500 rpm for 10 min. After centrifugation, take 100 μL of the supernatant and transfer it to a new 96-well plate, make marks, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 540 nm, and calculate the hemolysis rate. Hemolysis rate (%) = [(OD experimental group - OD negative control) / (OD positive control - OD negative control)] x 100%.

[0152] 2) Experimental results

[0153] The hemolysis rate results of penicillin G hydroxamic acid on red blood cells are as Figure 7As shown, under the action of 0.5% Triton X-100, the hemolysis rate of red blood cells reached 100%, showing a strong hemolytic effect. Even when the concentration of penicillin G hydroxamic acid reached 128 μg / mL, its hemolysis rate for red blood cells was 0, indicating no hemolysis. The above results show that penicillin G hydroxamic acid has no damaging ability to the red blood cell membrane and has high safety.

[0154] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. Penicillin G hydroxamic acid, characterized in that It has the following chemical structure:

2. Use of the penicillin G hydroxamic acid according to claim 1 as a metallo-β-lactamase inhibitor in the preparation of a drug for reversing bacterial resistance, wherein the bacteria are resistant bacteria expressing metallo-β-lactamases (MBLs).

3. The use according to claim 2, characterized in that: The metallo-β-lactamase is of NDM-1 or VIM-2 type.

4. Use of the penicillin G hydroxamic acid according to claim 1 as a metallo-β-lactamase inhibitor in the preparation of a drug for reversing bacterial resistance, or use of the penicillin G hydroxamic acid according to claim 1 as a metallo-β-lactamase inhibitor combined with β-lactam antibiotics in the preparation of a drug against resistant bacteria, wherein the bacteria are resistant bacteria expressing metallo-β-lactamase.

5. The use according to claim 4, characterized in that: The drug-resistant bacteria expressing metal β-lactamase are drug-resistant Gram-negative bacteria expressing metal β-lactamase; preferably, the drug-resistant bacteria expressing metal β-lactamase are drug-resistant Gram-negative brevicaurus expressing metal β-lactamase; more preferably, the drug-resistant bacteria expressing metal β-lactamase are drug-resistant Escherichia coli expressing metal β-lactamase.

6. The use according to claim 4, characterized in that: The β-lactam antibiotic is a carbapenem antibiotic; preferably the carbapenem antibiotic is meropenem.

7. An antibacterial combination drug, the active ingredients of which include the penicillin G hydroxamic acid and β-lactam antibiotics according to claim 1, the penicillin G hydroxamic acid and β-lactam antibiotics are respectively independent dosing units, or the penicillin G hydroxamic acid and β-lactam antibiotics together form a combined dosing unit, and the bacteria are resistant bacteria expressing metallo-β-lactamase.

8. The combined drug according to claim 7, characterized in that: The drug-resistant bacteria expressing metal β-lactamase are drug-resistant Gram-negative bacteria expressing metal β-lactamase; preferably, the drug-resistant bacteria expressing metal β-lactamase are drug-resistant Gram-negative brevicaurus expressing metal β-lactamase; more preferably, the drug-resistant bacteria expressing metal β-lactamase are drug-resistant Escherichia coli expressing metal β-lactamase.

9. The combined drug according to claim 7, characterized in that: The metallo-β-lactamase is of NDM-1 or VIM-2 type.

10. The combined drug according to any one of claims 7 to 9, characterized in that: The β-lactam antibiotic is a carbapenem antibiotic; preferably the carbapenem antibiotic is meropenem.

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