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Bacterial resistance caused by metallo‐β‐lactamases (MβLs) has become an emerging public health threat, and the development of MβLs inhibitor is an effective way to overcome the resistance. In this study, thirteen novel O‐aryloxycarbonyl hydroxamates were constructed and assayed against MβLs. The obtained molecules specifically inhibited imipenemase‐1 (IMP‐1) and New Delhi metallo‐β‐lactamase‐1, exhibiting an IC50 value in the range of 0.10–18.42 and 0.23–22.33 μM, respectively. The hydroxamate 5 was found to be the most potent inhibitor, with an IC50 of 0.1 and 0.23 μM using meropenem and cefazolin as substrates. ICP‐MS analysis showed that 5 did not coordinate to the Zn(II) ions at the active site of IMP‐1, while the rapid dilution, thermal shift and MALDI‐TOF assays revealed that the hydroxamate formed a covalent bond with the enzyme. Cytotoxicity assays indicated that the hydroxamates have low toxicity in MCF‐7 cells. This work provided a potent scaffold for the development of MβLs inhibitors. [ABSTRACT FROM AUTHOR]

Antibiotic resistance mediated by β-lactamases including metallo-β-lactamases (MβLs) has become an emerging threat. In this study, we employed UV-vis to monitor the activity and inhibition of New Delhi MβL (NDM), K. pneumoniae carbapenemase (KPC) and MβL VIM-2 in live bacterial cells in real time. The monitoring showed that the faropenem was hydrolyzed by six strains which express β-lactamases, K. pneumoniae, P. aeruginosa, E. coli expressing extended-spectrum β-lactamase (ESBL), NDM-1 (EC07), VIM-2, and methicillin-resistant S. aureus (MRSA), but not by E. coli and S. aureus which do not express β-lactamases. Also, four β-lactams cefazolin, meropenem, faropenem and amoxicillin were hydrolyzed by the NDM and KPC expressed in K. pneumoniae. Cell-based studies revealed that the targets NDM and KPC expressed in K. pneumoniae cells were inhibited by three known inhibitors ethylene diamine tetraacetic acid (EDTA), ebselen and clavulanic acid, with a fifty percent inhibitory concentration (IC50) of 1.2, 84.7 and 13.3 μM, and the VIM-2 expressed in E. coli cells was inhibited by EDTA, D -captopril and rhodanine with an IC50 of 1.3, 510.8 and 9.3 μM, respectively. This study provides an approach that could be applied to screen and evaluate small molecule inhibitors of β-lactamases in whole cells or to identify drug resistant bacteria. [ABSTRACT FROM AUTHOR]

[Display omitted] • New feature of Schiff base, inhibit enzyme that causing drug resistant, was found. • Compounds exhibited promising inhibition against metallo-β-lactamase. • Compounds restored antimicrobial activity of cefazolin against resistance bacteria. • From kinetics, thermodynamics and docking aspect, inhibit behavior was confirmed. • The most active compound was well tolerated with mouse fibroblast cells. The irregular use of antibiotics has created a natural selection pressure for bacteria to adapt resistance. Bacterial resistance caused by metallo-β-lactamases (MβLs) has been the most prevalent in terms of posing a threat to human health. The New Delhi metallo-β-lactamase-1 (NDM-1) has been shown to be capable of hydrolyzing almost all β-lactams. In this work, eight aromatic Schiff bases 1 – 8 were prepared and identified by enzyme kinetic assays to be the potent inhibitors of NDM-1 (except 4). These molecules exhibited a more than 95 % inhibition, and an IC 50 value in the range of 0.13–19 μM on the target enzyme, and 3 was found to be the most effective inhibitor (IC 50 = 130 nM). Analysis of structure–activity relationship revealed that the o -hydroxy phenyl improved the inhibitory activity of Schiff bases on NDM-1. The inhibition mode assays including isothermal titration calorimetry (ITC) disclosed that both compounds 3 and 5 exhibited a reversibly mixed inhibition on NDM-1, with a K i value of 1.9 and 10.8 μM, respectively. Antibacterial activity tests indicated that a dose of 64 μg·mL−1 Schiff bases resulted in 2–128-fold reduction in MICs of cefazolin on E. coli producing NDM-1 (except 4). Cytotoxicity assays showed that both Schiff bases 3 and 5 have low cytotoxicity on the mouse fibroblast (L929) cells at a concentration of up to 400 μM. Docking studies suggested that the hydroxyl group interacts with Gln123 and Glu152 of NDM-1, and the amino groups interact with the backbone amide groups of Glu152 and Asp223. This study provided a novel scaffold for the development of NDM-1 inhibitors. [ABSTRACT FROM AUTHOR]

[Display omitted] • N -acylhydrazones reversibly and competitively inhibit NDM-1; • N -acylhydrazones synergize meropenem to exhibit antibacterial effect in vivo and in vitro ; • N -acylhydrazones have low cytotoxicity. The expression of β-lactamases, especially metallo-β-lactamases (MβLs) in bacteria is one of the main causes of drug resistance. In this work, an effective N -acylhydrazone scaffold as MβL inhibitor was constructed and characterized. The biological activity assays indicated that the synthesized N -acylhydrazones 1 – 11 preferentially inhibited MβL NDM-1, and 1 was found to be the most effective inhibitor with an IC 50 of 1.2 µM. Analysis of IC 50 data revealed a structure–activity relationship, which is that the pyridine and hydroxylbenzene substituents at 2-position improved inhibition of the compounds on NDM-1. ITC and enzyme kinetics assays suggested that it reversibly and competitively inhibited NDM-1 (K i = 0.29 ± 0.05 µM). The synthesized N -acylhydrazones showed synergistic antibacterial activities with meropenem, reduced 4–16-fold MIC of meropenem on NDM-1- producing E. coli BL21 (DE3), while 1 restored 4-fold activity of meropenem on K. pneumonia expressing NDM-1 (NDM- K. pneumoniae). The mice experiments suggested that 1 combined meropenem to fight against NDM- K. pneumoniae infection in the spleen and liver. Cytotoxicity assays showed that 1 and 2 have low cytotoxicity. This study offered a new framework for the development of NDM-1 inhibitors. [ABSTRACT FROM AUTHOR]

• Diaryl-substituted thiosemicarbazones effective inhibit NDM-1. • 1c restored antibacterial effect of meropenem on bacteriaharboring NDM-1. • The inhibitor cooperated with meropenem to kill clinically resistant E.coli in vivo. The superbug infection caused by New Delhi metallo-β-lactamase (NDM-1) has become an emerging public health threat. Inhibition of NDM-1 has proven challenging due to its shuttling between pathogenic bacteria. A potent scaffold, diaryl-substituted thiosemicarbazone, was constructed and assayed with metallo-β-lactamases (MβLs). The obtained twenty-six molecules specifically inhibited NDM-1 with IC 50 0.038–34.7 µM range (except 1e , 2e , and 3d), and 1c is the most potent inhibitor (IC 50 = 0.038 µM). The structure-activity relationship of synthetic thiosemicarbazones revealed that the diaryl-substitutes, specifically 2-pyridine and 2-hydroxylbenzene improved inhibitory activities of the inhibitors. The thiosemicarbazones exhibited synergistic antimycobacterial actions against E. coli -NDM-1, resulted a 2–512-fold reduction in MIC of meropenem, while 1c restored 16–256-, 16-, and 2-fold activity of the antibiotic on clinical isolates ECs, K. pneumonia and P. aeruginosa harboring NDM-1, respectively. Also, mice experiments showed that 1c had a synergistic antibacterial ability with meropenem, reduced the bacterial load clinical isolate EC08 in the spleen and liver. This work provided a highly promising scaffold for the development of NDM-1 inhibitors. [ABSTRACT FROM AUTHOR]

The bacteria, harboring metallo-β-lactamases (MβLs), become resistant on most β-lactam antibiotics, specifically New Delhi metallo-β-lactamase-1 (NDM-1), which hydrolyzes almost all β-lactam antibiotics leading to bacterial multiple-drug resistance. It is highly desirable to develop effective NDM-1 inhibitors in reviving the efficacy of existing antibiotics. Here, we report a potent covalently reversible scaffold, 3-Bromopyruvate (3BP) to target the NDM-1 in vitro and in vivo. Enzymatic kinetic studies revealed that 3BP is capable of inhibiting the B1 and B2 MβLs and exhibited the best inhibition on NDM-1 with an IC 50 of 2.57 μM, also, it was found to be a dose- and time-dependent inhibitor. The study of inhibition mechanism suggested that 3BP reversibly inactivate NDM-1, and may form a dynamic reversible covalent bond with cysteine at active site of the enzyme. Besides, 3BP effectively restored the activity of five β-lactam antibiotics on three clinical strains expressing NDM-1, resulting in 2–8-fold reduction in MIC. Moreover, the toxicity evaluation of 3BP against L929 mouse fibroblastic cells indicated that 3BP had low cytotoxicity, implying it may be used as lead molecule for future drug candidate. Image, graphical abstract [ABSTRACT FROM AUTHOR]