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Metallo-β-lactamases (MBLs) are zinc-dependent enzymes capable of hydrolyzing all bicyclic β-lactam antibiotics, posing a great threat to public health. However, there are currently no clinically approved MBL inhibitors. Despite variations in their active sites, MBLs share a common catalytic mechanism with carbapenems, forming similar reaction species and hydrolysates. We here report the development of 2-aminothiazole-4-carboxylic acids (AtCs) as broad-spectrum MBL inhibitors by mimicking the anchor pharmacophore features of carbapenem hydrolysate binding. Several AtCs manifested potent activity against B1, B2, and B3 MBLs. Crystallographic analyses revealed a common binding mode of AtCs with B1, B2, and B3 MBLs, resembling binding observed in the MBL-carbapenem product complexes. AtCs restored Meropenem activity against MBL-producing isolates. In the murine sepsis model, AtCs exhibited favorable synergistic efficacy with Meropenem, along with acceptable pharmacokinetics and safety profiles. This work offers promising lead compounds and a structural basis for the development of potential drug candidates to combat MBL-mediated antimicrobial resistance.

The emergence of metallo-β-lactamases (MBLs) confers resistance to nearly all the β-lactam antibiotics, including carbapenems. Currently, there is a lack of clinically useful MBL inhibitors, making it crucial to discover new inhibitor chemotypes that can potently target multiple clinically relevant MBLs. Herein we report a strategy that utilizes a metal binding pharmacophore (MBP) click approach to identify new broad-spectrum MBL inhibitors. Our initial investigation identified several MBPs including phthalic acid, phenylboronic acid and benzyl phosphoric acid, which were subjected to structural transformations using azide-alkyne click reactions. Subsequent structure-activity relationship analyses led to the identification of several potent broad-spectrum MBL inhibitors, including 73 that manifested IC 50 values ranging from 0.00012 μM to 0.64 μM against multiple MBLs. Co-crystallographic studies demonstrated the importance of MBPs in engaging with the MBL active site anchor pharmacophore features, and revealed the unusual two-molecule binding modes with IMP-1, highlighting the critical role of flexible active site loops in recognizing structurally diverse substrates/inhibitors. Our work provides new chemotypes for MBL inhibition and establishes a MBP click-derived paradigm for inhibitor discovery targeting MBLs as well as other metalloenzymes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Resistance to β-lactam antibiotics is rapidly growing, substantially due to the spread of serine-β-lactamases (SBLs) and metallo-β-lactamases (MBLs), which efficiently catalyse β-lactam hydrolysis. Combinations of a β-lactam antibiotic with an SBL inhibitor have been clinically successful; however, no MBL inhibitors have been developed for clinical use. MBLs are a worrying resistance vector because they catalyse hydrolysis of all β-lactam antibiotic classes, except the monobactams, and they are being disseminated across many bacterial species worldwide. Here we review the classification, structures, substrate profiles, and inhibition mechanisms of MBLs, highlighting current clinical problems due to MBL-mediated resistance and progress in understanding and combating MBL-mediated resistance., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

As one of important mechanisms to β-lactam antimicrobial resistance, metallo-β-lactamases (MBLs) have been receiving increasing worldwide attentions. Ambler subclass B1 MBLs are most clinically relevant, because they can hydrolyze almost all β-lactams with the exception of monobactams. However, it is still lacking of clinically useful drugs to combat MBL-medicated resistance. We previously identified 1H-imidazole-2-carboxylic acid as a core metal-binding pharmacophore (MBP) to target multiple B1 MBLs. Herein, we report structural optimization of 1H-imidazole-2-carboxylic acid and substituents. Structure-activity relationship (SAR) analyses revealed that replacement of 1H-imidazole-2-carboxylic acid with other structurally highly similar MBPs excepting thiazole-4-carboxylic acid resulted in decreased MBL inhibition. Further SAR studies identified more potent inhibitors to MBLs, of which 28 manifested IC 50 values of 0.018 µM for both VIM-2 and VIM-5. The microbiological tests demonstrated that the most tested compounds showed improved synergistic effects; some compounds at 1 µg/ml were able to reduce meropenem MIC by at least 16-fold, which will be worth further development of new potent inhibitors particularly targeting VIM-type MBLs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Production of metallo-β-lactamases (MBLs) in bacterial pathogens is an important cause of resistance to the 'last-resort' carbapenem antibiotics. Development of effective MBL inhibitors to reverse carbapenem resistance in Gram-negative bacteria is still needed. We herein report X-ray structure-guided optimization of 1H-imidazole-2-carboxylic acid (ICA) derivatives by considering how to engage with the active-site flexible loops and improve penetration into Gram-negative bacteria. Structure-activity relationship studies revealed the importance of appropriate substituents at ICA 1-position to achieve potent inhibition to class B1 MBLs, particularly the Verona Integron-encoded MBLs (VIMs), mainly by involving ingenious interactions with the flexible active site loops as observed by crystallographic analyses. Of the tested ICA inhibitors, 55 displayed potent synergistic antibacterial activity with meropenem against engineered Escherichia coli strains and even intractable clinically isolated Pseudomonas aeruginosa producing VIM-2 MBL. The morphologic and internal structural changes of bacterial cells after treatment further demonstrated that 55 crossed the outer membrane and reversed the activity of meropenem. Moreover, 55 showed good pharmacokinetic and safety profile in vivo, which could be a potential candidate for combating VIM-mediated Gram-negative carbapenem resistance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

The production of β-lactamases represents the main cause of resistance to clinically important β-lactam antibiotics. Boron containing compounds have been demonstrated as promising broad-spectrum β-lactamase inhibitors to combat β-lactam resistance. Here we report a series of 3-aryl substituted benzoxaborole derivatives, which manifested broad-spectrum inhibition to representative serine-β-lactamases (SBLs) and metallo-β-lactamases (MBLs). The most potent inhibitor 9f displayed an IC 50 value of 86 nM to KPC-2 SBL and micromolar inhibitory activity towards other tested enzymes. Cell-based assays further revealed that 9f was able to significantly reduce the MICs of meropenem in clinically isolated KPC-2-producing bacterial strains and it showed no apparent toxicity in HEK293T cells., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Resistance to β-lactam antibacterials is commonly associated with the production of the serine β-lactamases (SBLs) and/or metallo-β-lactamases (MBLs). Although clinically useful inhibitors for the SBLs have been developed, no equivalent inhibitors are available for the MBLs, which can hydrolyze almost all β-lactam antibiotics, including the so-called "last resort" carbapenems. It is still a challenging task to develop a clinically useful inhibitor that should be broad-spectrum targeting multiple clinically relevant MBL enzymes that differ in their active site features. This review provides a detailed description of interaction modes of substrates and small-molecule inhibitors with various MBL enzymes and highlights the importance of metal- and "anchor residue"-binding features to achieve broad-spectrum MBL inhibition. Recently emerging active site interference strategies include metal ion deprivation, metal ion replacement, and cysteine modification as challenging, but worth experimenting directions for inhibitor development. The metalloenzyme selectivity, metal-binding pharmacophore, and cellular permeability and accumulation should be properly considered in the further development of clinically useful inhibitors to combat MBL-mediated antibacterial resistance., (© 2020 Wiley Periodicals, Inc.)

The emergence and spread of bacterial pathogens acquired metallo-β-lactamase (MBL) and serine-β-lactamase (SBL) medicated β-lactam resistance gives rise to an urgent need for the development of new dual-action MBL/SBL inhibitors. Application of a pharmacophore fusion strategy led to the identification of (2' S )-(1-(3'-mercapto-2'-methylpropanamido)methyl)boronic acid ( MS01 ) as a new dual-action inhibitor, which manifests broad-spectrum inhibition to representative MBL/SBL enzymes, including the widespread VIM-2 and KPC-2. Guided by the VIM-2: MS01 and KPC-2: MS01 complex structures, further structural optimization yielded new, more potent dual-action inhibitors. Selectivity studies indicated that the inhibitors had no apparent inhibition to human angiotensin-converting enzyme-2 and showed selectivity across serine hydrolyases in E. coli and human HEK293T cells labeled by the activity-based probe TAMRA-FP. Moreover, the inhibitors displayed potentiation of meropenem efficacy against MBL- or SBL-positive clinical isolates without apparent cytotoxicity. This work will aid efforts to develop new types of clinically useful dual-action inhibitors targeting MBL/SBL enzymes.

The emergence and global spread of metallo-β-lactamase (MBL) mediated resistance to almost all β-lactam antibacterials poses a serious threat to public health. Since no clinically useful MBL inhibitors have been reported, there is an urgent need to develop new potent broad-spectrum MBL inhibitors effective against antibacterial resistance. Herein, we synthesized a set of 2-substituted ((S)-3-mercapto-2-methylpropanamido) acetic acid derivatives, some of which displayed potent inhibition with high ligand efficiency to the clinically relevant MBL subtypes, Verona Integron-encoded MBL (VIM)-2 and New Delhi MBL (NDM)-1. Kinetic studies revealed that the inhibitors are not strong zinc chelators in solution, and they bind reversibly to VIM-2 but dissociate very slowly. Crystallographic analyses revealed that they inhibit VIM-2 via chelating the active site zinc ions and interacting with catalytically important residues. Further cell- and zebrafish-based assays revealed that the inhibitors slightly increase susceptibility of E. coli cells expressing VIM-2 to meropenem, and they have no apparent toxicity to the viability of HEK293T cells and the zebrafish embryogenesis., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Crystallographic analyses of the VIM-5 metallo-β-lactamase (MBL) with isoquinoline inhibitors reveal non zinc ion binding modes. Comparison with other MBL-inhibitor structures directed addition of a zinc-binding thiol enabling identification of potent B1 MBL inhibitors. The inhibitors potentiate meropenem activity against clinical isolates harboring MBLs.

Resistance to β-lactam antibiotics mediated by metallo-β-lactamases (MBLs) is a growing problem. We describe the use of protein-observe 19 F-NMR (PrOF NMR) to study the dynamics of the São Paulo MBL (SPM-1) from β-lactam-resistant Pseudomonas aeruginosa. Cysteinyl variants on the α3 and L3 regions, which flank the di-Zn II active site, were selectively 19 F-labeled using 3-bromo-1,1,1-trifluoroacetone. The PrOF NMR results reveal roles for the mobile α3 and L3 regions in the binding of both inhibitors and hydrolyzed β-lactam products to SPM-1. These results have implications for the mechanisms and inhibition of MBLs by β-lactams and non-β-lactams and illustrate the utility of PrOF NMR for efficiently analyzing metal chelation, identifying new binding modes, and studying protein binding from a mixture of equilibrating isomers., (© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)