Dipeptide compounds, pharmaceutical compositions and uses thereof

Abstract

The application relates to a dipeptide compound, a pharmaceutical composition and application thereof. The dipeptide compound has the structural characteristics shown in the following formula (1). The dipeptide compound can significantly improve the inhibitory activity of MBLs, and does not need to play an inhibitory role by chelating zinc ions, and has low toxic and side effects.

Description

Technical Field

[0001] This application relates to peptide compounds, and more particularly to a dipeptide compound, a pharmaceutical composition, and its application. Background Technology

[0002] In recent years, the overuse of antibiotics has led to serious bacterial resistance problems, posing a significant threat to global public health security. Beta-lactam antibiotics, due to their broad-spectrum antibacterial activity and good safety profile, have long been first-line drugs for clinical anti-infective therapy. However, with the continuous emergence of drug-resistant bacteria, their clinical efficacy has been severely challenged.

[0003] The production of metallo-β-lactamases (MBLs) is one of the main mechanisms leading to resistance to β-lactam antibiotics. MBLs can hydrolyze almost all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. Currently, there are no approved clinical inhibitors of metallo-β-lactamases (MBLIs, or MBL inhibitors) worldwide. This leaves patients infected with MBL-expressing resistant bacteria facing a lack of effective treatments. These resistant bacteria are also known as "superbugs."

[0004] Currently, the development of MBL inhibitors has become a global research hotspot. Among them, cysteine ​​inhibitors are considered highly promising candidate molecules due to the ability of their thiol groups to bind to zinc ions at the active sites of MBLs. For example, captopril, a reported cysteine-based MBL inhibitor, has a low therapeutic index and cannot meet clinical needs. Another approach involves linking cysteine ​​with other active amino acids to form dipeptide compounds. This is mainly achieved by forming a thioglycolic acid structure to shorten the distance between the thiol and carboxyl groups, and by using Fmoc to reduce polarity, thereby improving MBL inhibitory activity. However, the inhibitory activity of these dipeptide compounds still requires further improvement.

[0005] Meanwhile, most traditional MBL inhibitors exert their inhibitory effect by chelating zinc ions. These inhibitors often non-specifically chelate other metal ions in the body, leading to significant toxic side effects and limiting their potential as drugs. This is because some human metalloproteins belong to the MBL superfamily, and these non-specific chelators act on these proteins, causing severe toxic side effects. Furthermore, while many inhibitors exhibit good inhibitory activity at in vitro enzyme and bacterial levels, their efficacy in vivo is poor, failing to effectively restore the in vivo antibacterial activity of antibiotics. Summary of the Invention

[0006] Based on this, this application provides a dipeptide compound, a pharmaceutical composition, and its application that can significantly enhance the inhibitory activity of MBLs, exert its inhibitory effect without chelating zinc ions, and has low toxicity.

[0007] A first aspect of this application provides a dipeptide compound having the structural features shown in formula (1):

[0008] (1),

[0009] R1 is selected from amino protecting groups.

[0010] In one embodiment, R1 is selected from the Ac group or the Fmoc group.

[0011] In one embodiment, the dipeptide compound is selected from the following compounds:

[0012] .

[0013] A second aspect of this application provides the use of the dipeptide compounds described in the first aspect in the preparation of metallo-β-lactamase inhibitors.

[0014] A third aspect of this application provides the use of the dipeptide compounds described in the first aspect in the preparation of medicaments for improving resistance to β-lactam antibiotics.

[0015] In one embodiment, the β-lactam antibiotic is selected from meropenem.

[0016] A fourth aspect of this application provides a pharmaceutical composition comprising one or more dipeptide compounds as described in the first aspect, and a β-lactam antibiotic.

[0017] In one embodiment, the β-lactam antibiotic is selected from meropenem.

[0018] In one embodiment, the mass ratio of the dipeptide compound to the β-lactam antibiotic is (0.5~8):1.

[0019] A fifth aspect of this application provides the use of the pharmaceutical composition described in the fourth aspect in the preparation of a medicament having antibacterial properties.

[0020] This application, based on cysteine ​​research, discovered a novel peptide compound that significantly improves MBLs inhibitory activity, effectively restores the antibacterial activity of β-lactam antibiotics against MBL-producing resistant bacteria, and exhibits good in vivo therapeutic effects. This effectively addresses the current clinical dilemma of having no available MBL inhibitors, while avoiding the toxic side effects of nonspecific chelation of metal ions and demonstrating good biosafety.

[0021] Without limitation, the dipeptide compounds have the following advantages:

[0022] (1) The dipeptide compounds described herein exhibited strong in vitro inhibitory activity against clinically relevant metallo-β-lactamases (MBLs), including New Delhi metallo-β-lactamase-1 (NDM-1), Verona integron-encoded metallo-β-lactamase-2 (VIM-2), and imipenemase-1 (IMP-1), with inhibition constants (K0). i The values ​​are all in the low micromolar range and are superior to those of captopril previously reported.

[0023] (2) The dipeptide compounds have excellent biosafety, no obvious cytotoxicity or hemolytic activity, and no obvious toxic side effects in vivo. Moreover, their inhibition mechanism is competitive inhibition, which is different from traditional zinc ion deprivation inhibitors. Their inhibitory activity is not affected by exogenous zinc ions, thus avoiding the toxic side effects caused by non-specific chelation of metal ions. They have excellent drug development potential.

[0024] (3) Combining the dipeptide compound with β-lactam antibiotics can reduce the minimum inhibitory concentration (MIC) of β-lactam antibiotics against drug-resistant bacteria expressing MBLs, effectively restore the antibacterial activity of β-lactam antibiotics against drug-resistant bacteria, and the synergistic antibacterial index (FICI) between the two is small, indicating significant synergistic antibacterial activity.

[0025] (4) Combining the dipeptide compound with β-lactam antibiotics can significantly improve the survival rate of mice infected with drug-resistant bacteria expressing MBLs, up to 100%, and can effectively reduce the bacterial load of target organs and alleviate pathological damage to tissues, thus having a good in vivo therapeutic effect.

[0026] (5) The dipeptide compounds can be prepared by chemical synthesis, which is easy to synthesize, can be mass-produced, and is easy to promote and apply. Attached Figure Description

[0027] To better describe and illustrate the embodiments or examples provided in this application, reference may be made to one or more accompanying drawings. Additional details or examples used to describe the drawings should not be considered as limiting the scope of any of the disclosed applications, the currently described embodiments or examples, or the best mode of conduct of these applications as currently understood. In the drawings:

[0028] Figure 1 The diameter of the inhibition zone of Fmoc-MC and captopril combined with meropenem in paper discs against E. coli (NDM-1), E. coli (VIM-2), and E. coli (IMP-1).

[0029] Figure 2 The time-kill curves of Fmoc-MC and meropenem alone or in combination against E. coli (NDM-1) strain are shown.

[0030] Figure 3 The effect of the combination of Fmoc-MC and meropenem on the survival rate of infected mice.

[0031] Figure 4 The effect of the combination of Fmoc-MC and meropenem on the bacterial load of target organs in infected mice.

[0032] Figure 5 This is an HE staining experiment of target organs of mice infected after Fmoc-MC was combined with meropenem.

[0033] Figure 6 These are the results of routine blood and serum biochemical analysis of mice treated with Fmoc-MC.

[0034] Figure 7 Different concentrations of Fmoc-MC and EDTA were added to Zn 2+ The residual activity of NDM-1 afterward.

[0035] Figure 8 It is a Lineweaver-Burk plot of Fmoc-MC and EDTA.

[0036] Figure 9 This is a scanning electron microscope observation of the morphological changes of NDM-1-producing Escherichia coli cells after co-incubation with meropenem and Fmoc-MC (A is the blank control group, B and C represent meropenem and Fmoc-MC alone incubated with NDM-1-producing Escherichia coli, respectively, and D shows the results of incubation with NDM-1-producing Escherichia coli after combined incubation with meropenem and Fmoc-MC).

[0037] Figure 10 This is a schematic diagram of the interaction between Fmoc-MC and three MBLs: NDM-1, VIM-2, and IMP-1 (AC represents the molecular docking results of Fmoc-MC with NDM-1, VIM-2, and IMP-1). Detailed Implementation

[0038] The dipeptide compounds, pharmaceutical compositions, and their applications of this application are further described in detail below with reference to specific embodiments. This application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of this application.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0040] In this article, "one or more" refers to any one, two or more of the listed items.

[0041] In this application, terms such as "first aspect," "second aspect," "third aspect," and "fourth aspect" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, terms such as "first," "second," "third," and "fourth" serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0042] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0043] In this application, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0044] Unless otherwise specified, the percentage content mentioned in this application refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0045] Unless otherwise specified, all percentage concentrations mentioned in this application refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of that component.

[0046] Unless otherwise specified, the temperature parameters in this application may be either constant temperature processing or processing within a certain temperature range. The constant temperature processing allows for temperature fluctuations within the precision range controlled by the instrument.

[0047] In this application, room temperature generally refers to 4℃~30℃, and preferably 20±5℃.

[0048] Some embodiments of this application provide dipeptide compounds having the structural features shown in formula (1):

[0049] (1),

[0050] R1 is selected from amino protecting groups.

[0051] Without limitation, R1 can be selected from conventional amino protecting groups in the art, such as Ac groups or Fmoc groups. It is understood that an Ac group represents an acetyl group; and an Fmoc group represents 9-fluorenylmethoxycarbonyl. In some embodiments, R1 is selected from an Fmoc group.

[0052] Without limitation, the dipeptide compound is selected from the following compounds:

[0053] .

[0054] Other embodiments of this application provide the use of the peptide compounds described above in the preparation of metallo-β-lactamase inhibitors. These peptide compounds exhibit significantly improved MBLs inhibitory activity and good in vivo therapeutic effects, while avoiding the toxic side effects of non-specific metal ion chelation, thus demonstrating good biocompatibility.

[0055] Other embodiments of this application provide the use of the peptide compounds described above in the preparation of medicaments for improving resistance to β-lactam antibiotics. The peptide compounds can effectively restore the antibacterial activity of β-lactam antibiotics against MBL-producing bacteria, improve resistance to β-lactam antibiotics, and enhance or restore their antibacterial efficacy.

[0056] Without limitation, the β-lactam antibiotic is selected from one or more of penicillins, cephalosporins, cephamycins, carbapenems, and thiomycins. As an example, the β-lactam antibiotic is selected from one or more of meropenem, imipenem, biapenem, ceftazidime, and piperacillin.

[0057] In some embodiments, the β-lactam antibiotic is selected from meropenem.

[0058] Other embodiments of this application provide a pharmaceutical composition comprising one or more peptide compounds as described above, and a β-lactam antibiotic. The peptide compounds can effectively restore the antibacterial activity of β-lactam antibiotics against MBL-producing resistant bacteria, improve resistance to β-lactam antibiotics, and there is synergistic antibacterial activity between the peptide compounds and the β-lactam antibiotics; combining the two can achieve a superior antibacterial effect.

[0059] Without limitation, the β-lactam antibiotic is selected from one or more of penicillins, cephalosporins, cephamycins, carbapenems, and thiomycins. As an example, the β-lactam antibiotic is selected from one or more of meropenem, imipenem, biapenem, ceftazidime, and piperacillin.

[0060] In some embodiments, the β-lactam antibiotic is selected from meropenem.

[0061] In some embodiments, the mass ratio of the peptide compound to the β-lactam antibiotic is (0.5~8):1. Specifically, this mass ratio includes, but is not limited to: 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, or any range between the foregoing.

[0062] Other embodiments of this application also provide the use of the pharmaceutical composition in the preparation of a medicament with antibacterial properties.

[0063] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this application document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.

[0064] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art using known methods.

[0065] Example

[0066] This embodiment provides the following dipeptide compounds:

[0067] Fmoc-MC: .

[0068] The amino acid sequence of Fmoc-MC is Met-Cys, with the N-terminus modified by the Fmoc group. The synthesis method is as follows:

[0069] A solid-phase synthesis strategy using Fmoc was employed, with 2-chlorotriphenylmethyl chloride resin (2-Chlorotrityl Chloride Resin) as the solid-phase support, to sequentially couple Fmoc-protected amino acids. The degree of resin substitution was 0.84 mmol / g, and the synthesis scale was 2 g of resin.

[0070] Resin swelling: Weigh 2 g of resin and place it in a reaction tube, add dichloromethane (DCM, 15 mL / g resin), and shake for 30 min to allow the resin to swell fully.

[0071] First amino acid coupling: DCM was removed by filtration. 1.5 molar excess (relative to resin substitution) of Fmoc-Cys(Trt)-OH was added to the reaction tube, followed by 10 molar excess of N,N-diisopropylethylamine (DIEA), dissolved in a small amount of N,N-dimethylformamide (DMF). The mixture was shaken and reacted for 1 h. After coupling, the resin was washed 6 times alternately with DMF and DCM.

[0072] End-capping: Add an appropriate amount of methanol to the reaction tube to seal the unreacted active sites in order to avoid affecting the subsequent coupling efficiency.

[0073] Fmoc deprotection: Add 15 mL of 20% piperidine / DMF solution (v / v), shake for 5 min, and filter to remove the protection; add another 15 mL of 20% piperidine / DMF solution, shake for 15 min. After deprotection, wash the resin with DMF. Take a small amount of resin for ninhydrin colorimetric detection: Wash the resin three times with ethanol, add 1 drop each of ninhydrin, KCN, and phenol solution, and heat at 105–110℃ for 5 min. A deep blue color in the resin indicates a positive reaction (indicating free amino groups).

[0074] Amino acid coupling: Dissolve 3 molar excesses of Fmoc-Met-OH and 3 molar excesses of HBTU in a small amount of DMF, add the solution to a reaction tube, and immediately add 10 molar excesses of N-methylmorpholine (NMM). Shake and react for 30 min. After the reaction is complete, take a small amount of resin for ninhydrin colorimetric detection. A colorless or pale yellow resin indicates complete coupling.

[0075] Resin washing and drying: After coupling, the resin was washed once with DMF (10 mL / g), twice with methanol (10 mL / g), twice with DMF (10 mL / g), and twice with DCM (10 mL / g) in sequence, and finally dried until no solvent residue remained.

[0076] Peptide cleavage: A cleavage reagent was prepared with a composition of TFA / water / EDT / TIS = 94 : 2.5 : 2.5 : 1 (v / v / v / v). The cleavage reagent (10 mL / g resin) was added according to the resin mass, and the reaction was carried out at room temperature with shaking for 120 min. After the reaction, the lysis buffer was dried under a nitrogen stream, and cold diethyl ether was added to precipitate the crude peptide. The peptide was washed 6 times with diethyl ether and evaporated to dryness at room temperature to obtain the crude peptide product. The crude peptide was purified by reversed-phase HPLC, and the final purity reached over 95%. 1 H NMR, 13 The structure of the obtained polypeptide was confirmed by C10 NMR and electrospray ionization mass spectrometry (ESI-MS).

[0077] 1H NMR (600 MHz, DMSO-d6): δ (ppm) 7.89-7.77 (2H), 7.70-7.64 (2H), 7.44-7.32 (4H), 4.33-4.29 (2H), 4.27-4.23 (1H), 4.20-4.12 (1H), 4.10-4.07(1H), 3.64-3.57 (2H), 2.88-2.85 (2H), 2.50-2.45 (2H), 2.03 (3H). 13 C NMR (150MHz, DMSO-d6) δ / ppm: 179.49, 170.74, 156.03, 143.66, 140.71, 127.64, 127.12,125.33, 125.29, 120.09, 65.66, 55.30, 54.13, 46.68, 31.57, 26.61, 14.61. m / z475.35 [M+H] + 949.76 [2M+H] + .

[0078] Comparative Example

[0079] This comparative example provides the following dipeptide compounds:

[0080] Fmoc-WC: .

[0081] The amino acid sequence of Fmoc-WC is Trp-Cys, with the N-terminus modified by the Fmoc group. The synthesis method is similar to that in Example 1, except that Fmoc-Met is replaced with Fmoc-Trp.

[0082] Test Example 1

[0083] This test case demonstrates the in vitro MBLs inhibitory activity of various peptide compounds.

[0084] (1) Construction of recombinant engineered bacteria

[0085] Recombinant plasmids (NDM-1, VIM-2 and IMP-1) and E. coli BL21(DE3) were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0086] Remove BL21(DE3) competent cells from the -80℃ cryogenic freezer and thaw them on ice. In a clean bench, add 1 μL of recombinant plasmids (NDM-1, VIM-2, and IMP-1) to 100 μL of BL21(DE3) competent cells and gently pipette to mix. Then, incubate the competent cells on ice for 30 min, followed by heat shock at 42℃ for 90 s, and then on ice for another 2 min. Add 900 μL of LB (Luria-Bertani) liquid medium (without kanamycin) to the competent cells and incubate at 37℃ and 220 rpm for 1 h in a constant temperature shaking incubator. Spread the competent cell culture evenly onto LB solid medium containing 25 μg / mL kanamycin and incubate inverted mode overnight at 37℃.

[0087] (2) Isolation and purification of MBLs

[0088] 1) Solution preparation

[0089] LB medium: Weigh 12.5g of LB medium powder into a conical flask containing 500mL of ddH2O, dissolve it, autoclave and cool to room temperature before use.

[0090] Kanamycin-resistant LB agar medium: Weigh 12.5g of LB medium and 7.5g of agar powder into a conical flask containing 500mL of ddH2O and dissolve. Autoclave and cool to 55℃~60℃, then add 1250 μL of 10mg / mL kanamycin sulfate to the solution and mix well. Pour the mixture into sterile petri dishes and cool for later use.

[0091] Lysis buffer: Weigh 2.383g HEPES and 3.506g NaCl into an Erlenmeyer flask containing 200mL ddH2O, dissolve them, add 200μL Triton X-100 and mix well. Adjust the pH to approximately 8.0; that is, 50mM HEPES + 300mM NaCl + 0.1% Triton X-100, pH≈8.0.

[0092] Binding buffer: Weigh 2.383g HEPES and 3.506g NaCl into an Erlenmeyer flask containing 200mL ddH2O, dissolve them, add 400μL Triton X-100 and mix well. Adjust the pH to approximately 8.0; that is, 50mM HEPES + 300mM NaCl + 0.2% Triton X-100, pH≈8.0.

[0093] Washing buffer: Weigh 2.383g HEPES, 3.506g NaCl, 0.681g imidazole and 0.003g ZnCl2 into a 200mL Erlenmeyer flask containing ddH2O, dissolve them, and adjust the pH to approximately 8.0; that is, 50mM HEPES + 300mM NaCl + 50mM imidazole + 0.1mM ZnCl2, pH≈8.0.

[0094] Elution buffer: Weigh 2.383g HEPES, 3.506g NaCl, 3.404g imidazole and 0.003g ZnCl2 into an Erlenmeyer flask containing 200mL ddH2O, dissolve, and adjust the pH to approximately 8.0; that is, 50mM HEPES + 300mM NaCl + 250mM imidazole + 0.1mM ZnCl2, pH≈8.0.

[0095] HEPES buffer: Weigh 5.95 g of HEPES into 500 mL of ddH2O to a final concentration of 50 mM, and adjust the pH to approximately 7.0. Then add bovine serum albumin (BSA) to a final concentration of 50 µg / mL, dimethyl sulfoxide (DMSO) to a final concentration of 1.25%, and Triton X-100 to a final concentration of 0.01%.

[0096] Replacement buffer 1: Weigh 5.958g HEPES, 8.766g NaCl and 0.0068g ZnCl2 into an Erlenmeyer flask containing 500mL ddH2O, dissolve them, and adjust the pH to about 8.0; that is, 50mM HEPES + 300mM NaCl + 0.1mM ZnCl2, pH≈8.0.

[0097] Replacement buffer 2: Weigh 5.958g HEPES, 5.844g NaCl and 0.0068g ZnCl2 into an Erlenmeyer flask containing 500mL ddH2O, dissolve them, and adjust the pH to about 8.0; that is, 50mM HEPES + 200mM NaCl + 0.1mM ZnCl2, pH≈8.0.

[0098] Replacement buffer 3: Weigh 5.958g HEPES, 4.383g NaCl and 0.0068g ZnCl2 into an Erlenmeyer flask containing 500mL ddH2O, dissolve them, and adjust the pH to about 8.0; that is, 50mM HEPES + 150mM NaCl + 0.1mM ZnCl2, pH≈8.0.

[0099] 2) Expression and purification of three clinically common metallo-β-lactamases (NDM-1, VIM-2, IMP-1):

[0100] ① Expression and purification of NDM-1

[0101] Resuscitation: Take one loopful of glycerol bacteria carrying the NDM-1 gene plasmid using an inoculation loop, inoculate it onto an LB agar plate containing 25 μg / mL kanamycin, and incubate it overnight at 37°C with shaking for 12-16 hours.

[0102] Activation: Pick a single colony of Escherichia coli expressing NDM-1 with an inoculation loop and add it to 10 mL of LB liquid medium containing 25 μg / mL kanamycin. Incubate overnight at 37°C and 150 rpm for 12-16 h on a shaker.

[0103] Scale-up culture: Take 2.5 mL of the bacterial culture from the previous step and add it to 250 mL of LB liquid medium containing 25 μg / mL kanamycin (two portions). Incubate at 37°C and 150 rpm on a shaker for about 4 hours until the OD500 of the bacterial culture reaches the target concentration. 600 The value is 0.6~0.8.

[0104] Low-temperature induction culture: Add 250 μL of 0.1 M IPTG (isopropyl-β-D-thiogalactoside) to the above bacterial culture and induce culture at low temperature. Incubate overnight at 18℃ and 150 rpm for about 20 h on a shaker.

[0105] Cell collection: Centrifuge the low-temperature induced bacterial culture at 10,000 rpm at 4°C for 5 min. Discard the supernatant culture medium, resuspend the bacterial pellet in an appropriate amount of sterile PBS, centrifuge (10,000 rpm, 4°C, 5 min), remove the supernatant PBS, and store the bacterial pellet at -80°C.

[0106] Cell disruption: Resuspend the frozen cell pellet from the previous step with an appropriate amount of lysis buffer and add the corresponding volume of protease inhibitor (×100). Then, place the resuspended bacterial solution in an ice bath and sonicate it using a cell sonicator at 400W for 5 seconds with a 5-second interval, for a total of 15 minutes.

[0107] Centrifugation: Centrifuge the disrupted bacterial culture (12000 rpm, 4℃, 3 min). Collect the supernatant, and then filter the supernatant through a 0.45 μM microporous membrane to obtain crude protein. Also collect a small amount of bacterial precipitate for subsequent analysis.

[0108] 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. Next, wash the column with washing buffer containing 50 mM imidazole to remove contaminating proteins, for a total of 6 washes; then elute with elution buffer containing 250 mM imidazole, collect the eluent, and finally treat the nickel column and store it in 20% ethanol.

[0109] SDS-PAGE gel electrophoresis: The crude protein, eluent, and other samples collected in the previous step were subjected to SDS-PAGE gel electrophoresis, stained with Coomassie Brilliant Blue rapid staining solution, and the protein purification status was identified and collected.

[0110] Dialysis: The purified protein was placed in a dialysis bag and dialyzed for 2-4 hours with replacement buffer 1. Then, replacement buffer 2 was used for 6-8 hours, followed by dialysis with replacement buffer 3 for 12 hours. After 12 hours of dialysis, replacement buffer 3 was used again for 2 hours of dialysis. The dialyzed protein sample was collected and filtered through a 0.22 μm microporous membrane to collect the target protein. The collected target protein was then subjected to subsequent enzyme activity assays, aliquoted, and stored at -20°C.

[0111] ② Expression and purification of VIM-2

[0112] Resuscitation: Take one loopful of glycerol bacteria carrying the VIM-2 gene plasmid using an inoculation loop, inoculate it onto an LB agar plate containing 25 μg / mL kanamycin, and incubate it overnight at 37°C with shaking for 12-16 hours.

[0113] Activation: Pick a single colony of Escherichia coli expressing VIM-2 with an inoculation loop and add it to 10 mL of LB liquid medium containing 25 μg / mL kanamycin. Incubate overnight at 37°C and 150 rpm for 12-16 hours on a shaker.

[0114] Scale-up culture: Take 2.5 mL of the bacterial culture from the previous step and add it to 250 mL of LB liquid medium containing 25 μg / mL kanamycin (two portions). Incubate at 37°C and 150 rpm on a shaker for about 4 hours until the OD500 of the bacterial culture reaches the target concentration. 600 The value is 0.6~0.8.

[0115] Low-temperature induction culture: Add 250 μL of 0.1 M IPTG to the above bacterial culture and induce culture at low temperature. Incubate overnight at 18℃ and 150 rpm for about 20 h on a shaker.

[0116] Cell collection: Centrifuge the low-temperature induced bacterial culture (10,000 rpm, 4°C, 5 min). Discard the supernatant culture medium, resuspend the bacterial pellet in an appropriate amount of sterile PBS, centrifuge (10,000 rpm, 4°C, 5 min), remove the supernatant PBS, and store the bacterial pellet at -80°C.

[0117] Cell disruption: Resuspend the frozen cell pellet from the previous step with an appropriate amount of lysis buffer and add the corresponding volume of protease inhibitor (×100). Then, place the resuspended bacterial solution in an ice bath and sonicate it using a cell sonicator at 400W for 5 seconds with a 5-second interval, for a total of 15 minutes.

[0118] Centrifugation: Centrifuge the disrupted bacterial culture (12000 rpm, 4℃, 3 min). Collect the supernatant, and then filter the supernatant through a 0.45 μM microporous membrane to obtain crude protein. Also collect a small amount of bacterial precipitate for subsequent analysis.

[0119] 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. Next, wash the column with washing buffer containing 50 mM imidazole to remove contaminating proteins, for a total of 6 washes; then elute with elution buffer containing 250 mM imidazole, collect the eluent, and finally treat the nickel column and store it in 20% ethanol.

[0120] SDS-PAGE gel electrophoresis: The crude protein, eluent, and other samples collected in the previous step were subjected to SDS-PAGE gel electrophoresis, stained with Coomassie Brilliant Blue rapid staining solution, and the protein purification status was identified and collected.

[0121] Dialysis: The purified protein was placed in a dialysis bag and dialyzed for 2-4 hours with replacement buffer 1. Then, replacement buffer 2 was used for 6-8 hours, followed by dialysis with replacement buffer 3 for 12 hours. After 12 hours of dialysis, replacement buffer 3 was used again for 2 hours of dialysis. The dialyzed protein sample was collected and filtered through a 0.22 μm microporous membrane to collect the target protein. The collected target protein was then subjected to subsequent enzyme activity assays, aliquoted, and stored at -20°C.

[0122] 3) Expression and purification of IMP-1

[0123] Resuscitation: Take one loopful of glycerol bacteria carrying the IMP-1 gene plasmid using an inoculation loop, inoculate it onto an LB agar plate containing 25 μg / mL kanamycin, and incubate it overnight at 37°C with shaking for 12-16 hours.

[0124] Activation: Pick a single colony of Escherichia coli expressing IMP-1 with an inoculation loop and add it to 10 mL of LB liquid medium containing 25 μg / mL kanamycin. Incubate overnight at 37°C and 150 rpm for 12-16 h on a shaker.

[0125] Scale-up culture: Take 2.5 mL of the bacterial culture from the previous step and add it to 250 mL of LB liquid medium containing 25 μg / mL kanamycin (two portions). Incubate at 37°C and 150 rpm on a shaker for about 4 hours until the OD500 of the bacterial culture reaches the target concentration. 600 The value is 0.6~0.8.

[0126] Low-temperature induction culture: Add 250 μL of 0.1 M IPTG to the above bacterial culture and induce culture at low temperature. Incubate overnight at 18℃ and 150 rpm for about 20 h on a shaker.

[0127] Cell collection: Centrifuge the low-temperature induced bacterial culture (10,000 rpm, 4°C, 5 min). Discard the supernatant culture medium, resuspend the bacterial pellet in an appropriate amount of sterile PBS, centrifuge (10,000 rpm, 4°C, 5 min), remove the supernatant PBS, and store the bacterial pellet at -80°C.

[0128] Cell disruption: Resuspend the frozen cell pellet from the previous step in an appropriate amount of lysis buffer and add the corresponding volume of protease inhibitor (×100). Then, place the resuspended bacterial solution in an ice bath and sonicate it using a cell sonicator at 400W for 5 seconds with 5-second intervals, for a total of 15 minutes.

[0129] Centrifugation: Centrifuge the disrupted bacterial culture (12000 rpm, 4℃, 3 min). Collect the supernatant, and then filter the supernatant through a 0.45 μM microporous membrane to obtain crude protein. Also collect a small amount of bacterial precipitate for subsequent analysis.

[0130] 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. Next, wash the column with washing buffer containing 50 mM imidazole to remove contaminating proteins, for a total of 6 washes; then elute with elution buffer containing 250 mM imidazole, collect the eluent, and finally treat the nickel column and store it in 20% ethanol.

[0131] SDS-PAGE gel electrophoresis: The crude protein, eluent, and other samples collected in the previous step were subjected to SDS-PAGE gel electrophoresis, stained with Coomassie Brilliant Blue rapid staining solution, and the protein purification status was identified and collected.

[0132] Dialysis: The purified protein was placed in a dialysis bag and dialyzed for 2-4 hours with replacement buffer 1. Then, replacement buffer 2 was used for 6-8 hours, followed by dialysis with replacement buffer 3 for 12 hours. After 12 hours of dialysis, replacement buffer 3 was used again for 2 hours of dialysis. The dialyzed protein sample was collected and filtered through a 0.22 μm microporous membrane to collect the target protein. The collected target protein was then subjected to subsequent enzyme activity assays, aliquoted, and stored at -20°C.

[0133] (3) Enzyme inhibitory activity IC 50 Measurement

[0134] 1) Solution preparation

[0135] Cefotaxime working solution: Add 20 μL of 10 mM cefotaxime stock solution to 3.98 mL of HEPES buffer, mix well, and obtain a working solution with a final concentration of 0.05 mM.

[0136] Enzyme working solution: Add 100 μL of 1 μM enzyme stock solution to 400 μL of HEPES buffer, mix well, and obtain an enzyme working solution with a final concentration of 0.2 μM.

[0137] Captopril stock solution: Weigh an appropriate amount of captopril powder and dissolve it in HEPES buffer to a final captopril concentration of 20 mM. Store at -20°C for later use.

[0138] Peptide compound solution: Dissolve the peptide compound in DMSO to a final concentration of 20 mM, and store at -20℃ for later use.

[0139] 2) Enzyme kinetics experiment:

[0140] ① Experimental methods

[0141] Enzyme kinetics experiments were performed in 96-well plates. Cefotaxime was chosen as the hydrolysis substrate, and the detection wavelength was 492 nm. The final concentration of cefotaxime ranged from 1 μM to 30 μM. MBLs solution (NDM-1, VIM-2, and IMP-1 at a final concentration of 10 nM) was then added, and absorbance changes were immediately detected after mixing. Detection was repeated every 10 seconds for a total of 3 minutes. The hydrolysis rate in the first 30 seconds was used for K+ ionization. m The calculation.

[0142] ②Experimental Results

[0143] K of three MBLs (NDM-1, VIM-2 and IMP-1) m The value is consistent with existing reports and can be used for subsequent inhibitory activity assays.

[0144] 3) IC 50 Measurement:

[0145] ① Experimental methods

[0146] Dissolve each compound in sterile DMSO, then dilute with the appropriate buffer solution, ensuring the final DMSO concentration is below 2%. The compound concentration range is 0–300 μM, and the substrate cefotaxime concentration is 10 μM. Incubate the different concentrations of compounds with the enzyme for 15 min before adding the substrate to initiate the reaction. After mixing thoroughly, immediately measure the absorbance change at 492 nm. Measure every 10 s for 1 min, and use the increase in hydrolysis product in the first 30 s to calculate the initial reaction rate.

[0147] Calculate the residual activity of MBLs after adding different concentrations of inhibitors. Residual enzyme activity R(100%) = (V i / V0)×100%(V iV0: The rate of enzyme hydrolysis of substrate in the presence of inhibitor; V0: The rate of enzyme hydrolysis of substrate in the absence of inhibitor. The IC0 value was obtained by plotting a nonlinear fit between inhibitor concentration and residual enzyme activity using Graphpad Prism 8.0. 50 value.

[0148] ② Experimental Results

[0149] IC50 of each compound against three MBLs (NDM-1, VIM-2, and IMP-1) 50 The measurement results are shown in Table 1: Fmoc-MC showed strong inhibitory effects on NDM-1, VIM-2, and IMP-1, and the IC50 was obtained by fitting. 50 The values ​​are all relatively small, and the corresponding K i The values ​​were also at a low level, significantly better than the inhibitory activity of Fmoc-WC and the previously reported captopril.

[0150] Table 1. Inhibitory activity of peptide compounds against MBLs

[0151]

[0152] Test Example 2

[0153] This test case is an antibacterial experiment using peptide compounds in combination with β-lactam antibiotics.

[0154] (1) Chessboard Experiment

[0155] 1) Experimental methods

[0156] The experimental strain was a genetically engineered strain:

[0157] E. coli BL21 (DE3) / pET26b-NDM-1 is abbreviated as E. coli (NDM-1);

[0158] E. coli BL21 (DE3) / pET26b-VIM-2 is abbreviated as E. coli (VIM-2);

[0159] E. coli BL21 (DE3) / pET26b-IMP-1 is abbreviated as E. coli (IMP-1).

[0160] The MIC values ​​of meropenem alone and in combination with Fmoc-MC against three constructed strains were determined according to the microbroth dilution method established by the Clinical and Laboratory Standards Institute (CLSI). The combined antibacterial assay was performed as follows: The checkerboard microdilution method was used to evaluate the in vitro combined antibacterial effect of meropenem and the peptide compound. Meropenem was dissolved in sterile distilled water, and the peptide compound was dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions of 5120 μg / mL, which were then aliquoted and stored at -20°C for later use.

[0161] In 96-well plates, meropenem was serially diluted horizontally: 100 μL of 256 μg / mL meropenem solution was added to each well in column 1, and 50 μL of broth culture medium was pre-added to each well in columns 2 through 12. 50 μL of the solution was transferred from column 1 to column 2, mixed thoroughly, and then 50 μL was transferred to column 3. This serial dilution was repeated up to column 12. Finally, 50 μL was removed from column 12, resulting in a final volume of 50 μL per well. The final concentrations of meropenem were 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0 μg / mL, respectively.

[0162] Subsequently, the peptide compounds were serially diluted longitudinally: 50 μL of peptide compound solutions pre-diluted to different concentrations with culture medium were added to each well. The final concentrations of different rows (A to H) were 128, 64, 32, 16, 8, 4, 2 and 1 μg / mL, respectively, so that the concentrations of the two drugs in each well were arranged in a checkerboard pattern, and the final volume of each well was 100 μL.

[0163] Adjust the bacterial culture concentration to 0.5 McFarland concentration using culture medium, then dilute 100 times and add IPTG to a final concentration of 0.5 mM. Add 100 μL of the bacterial solution to the 96-well plate to achieve a final bacterial concentration of approximately 5 × 10⁻⁶ mM. 5 CFU / mL. Finally, the 96-well plates with added bacterial culture were incubated at 37°C for 16–20 h, and the MIC value was recorded. The MIC value is the minimum concentration of drug at which no obvious bacterial growth is observed with the naked eye.

[0164] Calculation of the synergistic antimicrobial index (FICI): The FICI value can be calculated according to a specific equation, i.e., FICI = FIC. A + FIC B FIC A The MIC value of drug A when used in combination with other drugs / the MIC value of drug A when used alone, FIC Bis defined as the MIC of drug B in combination divided by the MIC of drug B when used alone, and 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 significant interaction between the two drugs; if FICI > 4, it indicates that there is an antagonistic effect between the two drugs. The smaller the FICI index, the stronger the synergistic effect of the drugs.

[0165] 2) Experimental results

[0166] The results of FICI values of the combination of Fmoc-MC and meropenem against drug-resistant strains expressing NDM-1, VIM-2 and IMP-1 showed that the FICI of the combination of Fmoc-MC and meropenem against all three drug-resistant strains was ≤ 0.50, which indicated that this combination had good synergistic antibacterial activity against all three drug-resistant strains. The results are shown in Table 2. The combination can significantly reduce the MIC of meropenem against the above-mentioned drug-resistant strains and restore the antibacterial activity of meropenem.

[0167] Table 2 Minimum inhibitory concentration (MIC) of the combination of meropenem and peptide compound against genetically engineered MBL-expressing bacteria at a concentration of 128 μg / mL

[0168]

[0169] (2) Disk diffusion test (K-B method)

[0170] 1) Experimental method

[0171] Based on the checkerboard assay results, this experiment further verified the inhibitory effect of the combination of the peptide compound Fmoc-MC and meropenem on MBLs-producing constructed strains. With reference to the CLSI standards of the Clinical and Laboratory Standards Institute of the United States, the disk diffusion test was used to determine the size of the inhibition zone of meropenem alone, and the combination of meropenem with the peptide compound or the positive control drug captopril against the constructed strains.

[0172] First, meropenem susceptibility testing discs were prepared. Filter paper was punched into discs with a diameter of approximately 6 mm using a puncher, and then autoclaved and dried for later use. Discs containing 10 μg of meropenem were prepared according to CLSI standards, sealed, and stored at 4°C. Subsequently, a single colony of *E. coli* was picked and inoculated into LB medium containing kanamycin (final concentration 25 μg / mL). After incubation at 37°C and 150 rpm for 4 hours, the bacterial suspension was diluted to an OD600 value of 0.1. 100 μL of the bacterial suspension was dropped onto a sterile kanamycin-LB agar plate, and the suspension was evenly spread using a sterile cotton swab. The strain number and compound information were labeled. Using sterile forceps, a meropenem disc was precisely placed in the center of the agar plate, and 10 μL of different concentrations of inhibitor (0, 8, 16, 32 μg / mL) were added. After incubation at 37°C for 16–18 hours, the diameter of the inhibition zone was measured and recorded using calipers.

[0173] 2) Experimental Results

[0174] The experimental results were interpreted according to the CLSI standard: an inhibition zone diameter >15 mm indicated high sensitivity to the strain; an inhibition zone diameter of 10 mm to 15 mm indicated moderate sensitivity. The results for inhibition zone diameter are shown below. Figure 1 Compared with meropenem alone, the diameter of the inhibition zone was >15mm when meropenem was used in combination with compound Fmoc-MC, indicating that compound Fmoc-MC can restore the sensitivity of meropenem to the MBL-producing strains.

[0175] (3) Time-sterilization curve:

[0176] 1) Experimental methods

[0177] The constructed strain *E. coli* BL21(DE3) / pET26b-NDM-1 was used as the experimental strain. Meropenem (32 μg / mL), Fmoc-MC (32 μg / mL), combined drug group (32 μg / mL meropenem + 32 μg / mL Fmoc-MC), and a blank control group (containing only culture medium and bacterial suspension) were set up. Four sterile shaking tubes were used, and 10 mL of LB liquid medium containing the corresponding drug concentration was added to each tube. 100 µL of 0.5 McFarland concentration bacterial suspension was added to 10 mL of LB liquid medium (25 μg / mL kanamycin), and cultured at 37°C until the logarithmic growth phase (approximately 4-6 hours). Subsequently, the bacterial suspension concentration in the logarithmic growth phase was adjusted to 1.0 McFarland concentration using LB liquid medium, and 100 µL of bacterial suspension was added to each shaking tube. In 96-well plates, each bacterial culture was serially diluted 10-fold using LB liquid medium. 10 μL of each dilution was dropped onto MHA (Mueller-Hinton agar) plates. After incubating for approximately 30 minutes, the plates were inverted and incubated overnight at 37°C for 16–20 hours. The number of colonies on the plates was observed and recorded. Plotting time on the x-axis, logarithmic... 10 Plot a curve with CFU / mL as the ordinate.

[0178] 2) Experimental Results

[0179] The results of the time-sterilization curve experiment are as follows: Figure 2 As shown, the bacterial strains in the blank control group and the drug-free group grew normally, and neither 32 μg / mL meropenem nor 32 μg / mL Fmoc-MC alone could inhibit bacterial growth. However, when 32 μg / mL meropenem was used in combination with 32 μg / mL Fmoc-MC, the bacterial growth decreased significantly by 5 log within 8 hours. 10 The CFU / mL result indicates that Fmoc-MC can effectively restore the activity of meropenem against drug-resistant strains.

[0180] Test Example 3

[0181] This test case is an in vivo antibacterial efficacy experiment.

[0182] (1) Experimental methods

[0183] A systemic infection model was established in mice. Male BALB / c mice aged 6-8 weeks were randomly divided into 5 groups, with 6 mice in each group: blank control group, infection model group, meropenem monotherapy group, Fmoc-MC monotherapy group, and Fmoc-MC + meropenem combination therapy group.

[0184] Mice in the infected group were injected intraperitoneally with 5 × 10⁵ mg / L of the vaccine. 7A systemic infection model was established using CFU-producing NDM-1 Escherichia coli. One hour post-infection, mice in each group were treated with either meropenem (40 mg / kg) or Fmoc-MC (80 mg / kg) as single-drug groups, meropenem (40 mg / kg) combined with Fmoc-MC (80 mg / kg) as combined groups, and the model group received an equal volume of saline. Mice were observed for 7 consecutive days, and their survival was recorded. Forty-eight hours post-infection, some mice were sacrificed, and liver, spleen, and kidney tissues were harvested. The bacterial load in these tissues was measured, and HE staining was performed to observe histopathological changes.

[0185] (2) Experimental results

[0186] Survival results as follows Figure 3 The results showed that all mice in the infection model group died within 48 hours after infection. The survival rates of mice in the meropenem monotherapy group and the Fmoc-MC monotherapy group were 10% and 0%, respectively, while the survival rate of mice in the Fmoc-MC + meropenem combination therapy group was 90%, indicating that combination therapy can significantly improve the survival rate of infected mice. Bacterial load experiment results are as follows... Figure 4 The results showed that, compared with the infection model group, there were no significant changes in the bacterial load of target organs in infected mice before and after meropenem and Fmoc-MC monotherapy (P>0.05), indicating that they had no significant therapeutic effect. Meropenem combined with Fmoc-MC significantly reduced the bacterial load in the liver, spleen, and kidneys, with statistically significant differences (P<0.001). Pathological staining results are as follows: Figure 5 The results showed that the combined medication significantly reduced pathological damage to the liver, kidneys, and spleen of infected mice and improved tissue inflammation.

[0187] Test Example 4

[0188] This test case is a safety evaluation experiment.

[0189] (1) Experimental methods

[0190] The toxicity of Fmoc-MC to HEK-293T and HUVECs cells was detected by the CCK-8 assay. Cells were seeded in 96-well plates and cultured for 24 h. Different concentrations of the compound were added and cultured for another 24 h before cell viability was detected.

[0191] Red blood cell hemolysis experiment: Red blood cells were collected from mice, a red blood cell suspension was prepared, different concentrations of compounds were added, and after incubation for 1 h, the absorbance of the supernatant was detected and the hemolysis rate was calculated.

[0192] In vivo toxicity test: Mice were divided into a control group and a treatment group. The treatment group was given Fmoc-MC (80 mg / kg) for 7 consecutive days. Blood routine and blood biochemical indicators were detected, and major organs were stained with HE to observe toxicity.

[0193] (2) Experimental results

[0194] Cytotoxicity assays showed that Fmoc-MC at a concentration of 100 μM had no significant effect on the viability of HEK-293T and HUVECs cells, indicating that its cytotoxicity was extremely low.

[0195] The hemolysis test results showed that at a concentration of 100 μM, the hemolysis rate of the compound was less than 5%, indicating that it caused very little damage to the red blood cell membrane.

[0196] In vivo toxicity test results as follows Figure 6 The results showed that there were no significant differences in blood routine and blood biochemical indicators between the treated mice and the control group, and no obvious damage was found in the pathological sections of the major organs, indicating that the compound has good in vivo safety.

[0197] Test Example 5

[0198] This test case is for studying the mechanism of action.

[0199] (1) Experimental methods

[0200] Zinc ion recovery experiment: Different concentrations of exogenous Zn were added to the enzyme inhibition reaction system. 2+ The study aimed to determine whether the inhibitory activity of the compound against NDM-1 was affected, with EDTA used as a control.

[0201] Lineweaver-Burk curves: These curves are plotted to determine the type of inhibition by measuring the enzyme reaction rate at different substrate concentrations and the presence of different concentrations of compounds.

[0202] Scanning electron microscopy: NDM-1-producing Escherichia coli were treated with physiological saline, meropenem (32 μg / mL), Fmoc-MC (32 μg / mL), and meropenem (32 μg / mL) + Fmoc-MC (32 μg / mL), respectively. After fixation, the bacteria were observed under a scanning electron microscope to observe morphological changes.

[0203] Molecular docking: The binding mode of the compound with NDM-1 is simulated by molecular docking to analyze the binding site and interaction.

[0204] (2) Experimental results

[0205] The results of the zinc ion recovery experiment are as follows: Figure 7The results showed that the inhibitory activity of EDTA increased with the addition of exogenous Zn. 2+ The addition of Zn significantly reduced the activity of Fmoc-MC, while its inhibitory activity was unaffected by exogenous Zn. 2+ The influence indicates that its inhibitory mechanism is related to Zn 2+ Deprivation is irrelevant.

[0206] The Lineweaver-Burk curve results are as follows: Figure 8 The addition of the compound showed that the enzyme's K... m The value increases, while V max The unchanged result indicates that it is a competitive inhibition type, consistent with previous experimental results.

[0207] Scanning electron microscopy results as follows Figure 9 The results showed that the combined use of meropenem and Fmoc-MC caused morphological changes on the surface of NDM-1-producing Escherichia coli, including wrinkling, indentation, and rupture. This indicates that the combined use of drugs disrupted the bacterial cell membrane, causing the bacterial contents to leak out and exert their antibacterial effect.

[0208] Molecular docking analysis, such as Figure 10 This indicates that the compound Fmoc-MC binds to the active site of NDM-1. The binding mechanism is unrelated to zinc ions; it mainly binds to the enzyme through hydrogen bonds and hydrophobic interactions, thereby competitively blocking the binding of the substrate to the enzyme.

[0209] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0210] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. It should be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A dipeptide compound, characterized in that, It has the structural features shown in equation (1): （1）， R1 is selected from amino protecting groups.

2. The dipeptide compound according to claim 1, characterized in that, R1 is selected from Ac group or Fmoc group.

3. The dipeptide compound according to claim 1 or 2, characterized in that, The dipeptide compounds are selected from the following compounds: 。 4. The use of the dipeptide compound according to any one of claims 1 to 3 in the preparation of metallo-β-lactamase inhibitors.

5. The use of the dipeptide compound according to any one of claims 1 to 3 in the preparation of a medicament for improving resistance to β-lactam antibiotics.

6. The application according to claim 5, characterized in that, The β-lactam antibiotic is selected from meropenem.

7. A pharmaceutical composition, characterized in that, It includes one or more of the dipeptide compounds described in any one of claims 1 to 3, as well as β-lactam antibiotics.

8. The pharmaceutical composition according to claim 7, characterized in that, The β-lactam antibiotic is selected from meropenem.

9. The pharmaceutical composition according to claim 7 or 8, characterized in that, The mass ratio of the dipeptide compound to the β-lactam antibiotic is (0.5~8):

1.

10. Use of the pharmaceutical composition according to any one of claims 7 to 9 in the preparation of a medicament having antibacterial effects.

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