Cross-class metallo-β-lactamase inhibition by bisthiazolidines reveals multiple binding modes
28 Jun 2016-Proceedings of the National Academy of Sciences of the United States of America (National Academy of Sciences)-Vol. 113, Iss: 26, pp 201601368
TL;DR: A series of small compounds, bisthiazolidines, which act as inhibitors of all MBL types, restoring the efficacy of currently used antibiotics against resistant bacterial strains producing different MBLs are shown.
Abstract: Metallo-β-lactamases (MBLs) hydrolyze almost all β-lactam antibiotics and are unaffected by clinically available β-lactamase inhibitors (βLIs). Active-site architecture divides MBLs into three classes (B1, B2, and B3), complicating development of βLIs effective against all enzymes. Bisthiazolidines (BTZs) are carboxylate-containing, bicyclic compounds, considered as penicillin analogs with an additional free thiol. Here, we show both l- and d-BTZ enantiomers are micromolar competitive βLIs of all MBL classes in vitro, with Kis of 6-15 µM or 36-84 µM for subclass B1 MBLs (IMP-1 and BcII, respectively), and 10-12 µM for the B3 enzyme L1. Against the B2 MBL Sfh-I, the l-BTZ enantiomers exhibit 100-fold lower Kis (0.26-0.36 µM) than d-BTZs (26-29 µM). Importantly, cell-based time-kill assays show BTZs restore β-lactam susceptibility of Escherichia coli-producing MBLs (IMP-1, Sfh-1, BcII, and GOB-18) and, significantly, an extensively drug-resistant Stenotrophomonas maltophilia clinical isolate expressing L1. BTZs therefore inhibit the full range of MBLs and potentiate β-lactam activity against producer pathogens. X-ray crystal structures reveal insights into diverse BTZ binding modes, varying with orientation of the carboxylate and thiol moieties. BTZs bind the di-zinc centers of B1 (IMP-1; BcII) and B3 (L1) MBLs via the free thiol, but orient differently depending upon stereochemistry. In contrast, the l-BTZ carboxylate dominates interactions with the monozinc B2 MBL Sfh-I, with the thiol uninvolved. d-BTZ complexes most closely resemble β-lactam binding to B1 MBLs, but feature an unprecedented disruption of the D120-zinc interaction. Cross-class MBL inhibition therefore arises from the unexpected versatility of BTZ binding.
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TL;DR: It is contended that issues including continuing unresolved questions around mechanism; opportunities afforded by new technologies such as serial femtosecond crystallography; the need for new inhibitors, particularly for MBLs; the likely impact of new β-lactam:inhibitor combinations and the continuing clinical importance of β- lactams mean that this remains a rewarding research area.
423 citations
TL;DR: The authors show that the anti-Helicobacter pylori drug colloidal bismuth subcitrate inhibits MBLs by displacing the zinc ions with Bi(III), which is of great interest for the development of antibiotics.
Abstract: Drug-resistant superbugs pose a huge threat to human health. Infections by Enterobacteriaceae producing metallo-β-lactamases (MBLs), e.g., New Delhi metallo-β-lactamase 1 (NDM-1) are very difficult to treat. Development of effective MBL inhibitors to revive the efficacy of existing antibiotics is highly desirable. However, such inhibitors are not clinically available till now. Here we show that an anti-Helicobacter pylori drug, colloidal bismuth subcitrate (CBS), and related Bi(III) compounds irreversibly inhibit different types of MBLs via the mechanism, with one Bi(III) displacing two Zn(II) ions as revealed by X-ray crystallography, leading to the release of Zn(II) cofactors. CBS restores meropenem (MER) efficacy against MBL-positive bacteria in vitro, and in mice infection model, importantly, also slows down the development of higher-level resistance in NDM-1-positive bacteria. This study demonstrates a high potential of Bi(III) compounds as the first broad-spectrum B1 MBL inhibitors to treat MBL-positive bacterial infection in conjunction with existing carbapenems.
162 citations
TL;DR: This review will cover the evolution of currently available β-lactamase inhibitors along with the most recent research leading to new β- lactam enzyme inhibitors of potential clinical interest or already in the stage of clinical development.
146 citations
TL;DR: A new class of inhibitors for NDM-1 and two related β-lactamases, IMP and VIM-2, was identified by utilization of fragment-based drug discovery (FBDD), and was identified to be highly selective for MBLs when compared to other Zn(II) metalloenzymes.
Abstract: The efficacy of β-lactam antibiotics is threatened by the emergence and global spread of metallo-β-lactamase-(MBL) mediated resistance, specifically New Delhi-Metallo-β- lactamase-1 (NDM-1). Utilizing fragment-based drug discovery (FBDD), a new class of inhibitors for NDM-1 and two related β-lactamases, IMP-1 and VIM-2, was identified. Based on 2,6-dipicolinic acid (DPA), several libraries were synthesized for structure-activity relationship (SAR) analysis. Inhibitor 36 (IC50 = 80 nM) was identified to be highly selective for MBLs when compared to other Zn(II) metalloenzymes. While DPA displayed a propensity to chelate metal ions from NDM-1, 36 formed a stable NDM-1:Zn(II):inhibitor ternary complex, as demonstrated by 1H NMR, electron paramagnetic resonance (EPR) spectroscopy, equilibrium dialysis, intrinsic tryptophan fluorescence emission, and UV-Vis spectroscopy. When co-administered with 36 (at concentrations non-toxic to mammalian cells), the minimum inhibitory concentration (MIC) of imipenem against...
115 citations
TL;DR: Owing to their unique structure and function and their diversity, MBLs pose a particular challenge for drug development, although several stable agents and inhibitor combinations are at various stages in the development pipeline.
Abstract: Modern medicine is threatened by the global rise of antibiotic resistance, especially among Gram-negative bacteria. Metallo-β-lactamase (MBL) enzymes are a particular concern and are increasingly disseminated worldwide, though particularly in Asia. Many MBL producers have multiple further drug resistances, leaving few obvious treatment options. Nonetheless, and more encouragingly, MBLs may be less effective agents of carbapenem resistance in vivo, under zinc limitation, than in vitro Owing to their unique structure and function and their diversity, MBLs pose a particular challenge for drug development. They evade all recently licensed β-lactam-β-lactamase inhibitor combinations, although several stable agents and inhibitor combinations are at various stages in the development pipeline. These potential therapies, along with the epidemiology of producers and current treatment options, are the focus of this review.
115 citations
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TL;DR: CCP4mg is a project that aims to provide a general-purpose tool for structural biologists, providing tools for X-ray structure solution, structure comparison and analysis, and publication-quality graphics.
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27,505 citations
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18,531 citations
"Cross-class metallo-β-lactamase inh..." refers methods in this paper
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...Sfh-I crystals were pseudomerohedrally twinned [twin law h,-k,-l, determined by Xtriage (54)], so the Sfh-I model was completed with twin refinement in Refmac5....
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TL;DR: A description is given of Phaser-2.1: software for phasing macromolecular crystal structures by molecular replacement and single-wavelength anomalous dispersion phasing.
Abstract: Phaser is a program for phasing macromolecular crystal structures by both molecular replacement and experimental phasing methods. The novel phasing algorithms implemented in Phaser have been developed using maximum likelihood and multivariate statistics. For molecular replacement, the new algorithms have proved to be significantly better than traditional methods in discriminating correct solutions from noise, and for single-wavelength anomalous dispersion experimental phasing, the new algorithms, which account for correlations between F+ and F−, give better phases (lower mean phase error with respect to the phases given by the refined structure) than those that use mean F and anomalous differences ΔF. One of the design concepts of Phaser was that it be capable of a high degree of automation. To this end, Phaser (written in C++) can be called directly from Python, although it can also be called using traditional CCP4 keyword-style input. Phaser is a platform for future development of improved phasing methods and their release, including source code, to the crystallographic community.
17,755 citations
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TL;DR: The likelihood function for macromolecular structures is extended to include prior phase information and experimental standard uncertainties and the results derived are consistently better than those obtained from least-squares refinement.
Abstract: This paper reviews the mathematical basis of maximum likelihood The likelihood function for macromolecular structures is extended to include prior phase information and experimental standard uncertainties The assumption that different parts of a structure might have different errors is considered A method for estimating σA using `free' reflections is described and its effects analysed The derived equations have been implemented in the program REFMAC This has been tested on several proteins at different stages of refinement (bacterial α-amylase, cytochrome c′, cross-linked insulin and oligopeptide binding protein) The results derived using the maximum-likelihood residual are consistently better than those obtained from least-squares refinement
14,622 citations
"Cross-class metallo-β-lactamase inh..." refers methods in this paper
...Sfh-I crystals were pseudomerohedrally twinned [twin law h,-k,-l, determined by Xtriage (54)], so the Sfh-I model was completed with twin refinement in Refmac5....
[...]
...Structures were completed by iterative rounds of manual model building in Coot (53) and refinement in Phenix (54) or Refmac5 (55)....
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