A close look onto structural models and primary ligands of metallo-β-lactamases

doi:10.1016/j.drup.2018.08.001

Drug Resistance Updates

Volume 40, September 2018, Pages 1-12

https://doi.org/10.1016/j.drup.2018.08.001 Get rights and content

Abstract

β-Lactamases are hydrolytic enzymes capable of opening the β-lactam ring of antibiotics such as penicillin, thus endowing the bacteria that produce them with antibiotic resistance. Of particular medical concern are metallo-β-lactamases (MBLs), with an active site built around coordinated Zn cations. MBLs are pan-reactive enzymes that can break down almost all classes of β-lactams, including such last-resort antibiotics as carbapenems. They are not only broad-spectrum-reactive but are often plasmid-borne (e.g., the New Delhi enzyme, NDM), and can spread horizontally even among unrelated bacteria. Acquired MBLs are encoded by mobile genetic elements, which often include other resistance genes, making the microbiological situation particularly alarming. There is an urgent need to develop MBL inhibitors in order to rescue our antibiotic armory. A number of such efforts have been undertaken, most notably using the 3D structures of various MBLs as drug-design targets. Structure-guided drug discovery depends on the quality of the structures that are collected in the Protein Data Bank (PDB) and on the consistency of the information in dedicated β-lactamase databases. We conducted a careful review of the crystal structures of class B β-lactamases, concluding that the quality of these structures varies widely, especially in the regions where small molecules interact with the macromolecules. In a number of examples the interpretation of the bound ligands (e.g., inhibitors, substrate/product analogs) is doubtful or even incorrect, and it appears that in some cases the modeling of ligands was not supported by electron density. For ten MBL structures, alternative interpretations of the original diffraction data could be proposed and the new models have been deposited in the PDB. In four cases, these models, prepared jointly with the authors of the original depositions, superseded the previous deposits. This review emphasizes the importance of critical assessment of structural models describing key drug design targets at the level of the raw experimental data. Since the structures reviewed here are the basis for ongoing design of new MBL inhibitors, it is important to identify and correct the problems with ambiguous crystallographic interpretations, thus enhancing reproducibility in this highly medically relevant area.

Section snippets

Introduction – reproducibility crisis in biomedical research

A number of recent reports have brought to the attention of the scientific community the uncomfortable fact that a noticeable fraction of biomedical research cannot be reproduced (Minor et al., 2016; Prinz et al., 2011). This is an alarming trend, and even more alarming outlook, calling for immediate action. Not only are large resources of time, manpower, and money (estimated at $28 billion a year in the US alone (Freedman et al., 2015)) invested in potentially useless endeavors, but also false

Model validation methods

No new experimental data were collected for this work and all analyses were based on data obtained by others and deposited in the PDB and/or https://proteindiffraction.org/. We reviewed over 150 deposited crystal structures of metallo-β-lactamases and we used the BLDB database (Naas et al., 2017) to identify the relevant structures of B1, B2, and B3 β-lactamases. Only the entries containing experimental diffraction data were subjected to our detailed review.

When available, electron density maps

NDM-1 with tiopronin (PDB ID 5a5z)

A 2.6 Å structure of NDM-1, supposedly with the ligand tiopronin bound at the active site, was determined as part of a study entitled “Approved drugs containing thiols as inhibitors of metallo-β-lactamases: Strategy to combat multidrug-resistant bacteria” (Klingler et al., 2015). When this work was initially published, there was no accompanying PDB deposit. After our correspondence with the author and, subsequently, with the journal editor, the authors submitted the model coordinates and the

The ripple effect of sub-optimal and incorrect structural models

Crystallographic structural data, once deposited in the PDB and reported in the literature, tend to be treated by the non-structural community with full trust and their correctness is rarely questioned. The “ripple effect” of structural models (good and bad!) is very significant, given that many other fields of science use these models as the foundation for their research. Protein crystallography gives scientists an unprecedented power to investigate life at sub-microscopic levels, by revealing

Acknowledgments

We wish to thank Dr. Bernhard Rupp for valuable comments on one of the cases (5a5z). We also acknowledge, with thanks, the cooperation and joint authorship of the revised PDB deposits on the part of some of the original authors, especially Dr. Andrzej Joachimiak, Dr. Youngchang Kim, Dr. Natalie Strynadka, Dr. Dustin King, Dr. Alejandro Vila, Dr. Javier Gonzalez, Dr. Stefano Mangani, Dr. Isabel Garcia-Saez, Dr. Jean-Marie Frere, and Dr. Otto Dideberg. The work of MJ and JR was supported by the

References (52)

J.-D. Docquier et al.
An update on β-lactamase inhibitor discovery and development
Drug Resist. Update
(2018)
I. García-Sáez et al.
Three-dimensional structure of FEZ-1, a monomeric subclass B3 metallo-β-lactamase from Fluoribacter gormanii, in native form and in complex with D-captopril
J. Mol. Biol.
(2003)
M.L. Greenlee et al.
Synthesis and SAR of thioester and thiol inhibitors of IMP-1 metallo-β-lactamase
Bioorganic Med. Chem. Lett.
(1999)
W. Minor et al.
Safeguarding structural data repositories against bad apples
Structure
(2016)
Z. Otwinowski et al.
Processing of X-ray diffraction data collected in oscillation mode
Methods Enzymol.
(1997)
R.J. Read et al.
A new generation of crystallographic validation tools for the Protein Data Bank
Structure
(2011)
P.D. Adams et al.
PHENIX: a comprehensive Python-based system for macromolecular structure solution
Acta Crystallogr. Sect. D Biol. Crystallogr.
(2010)
H.M. Berman et al.
The protein data bank
Nucleic Acids Res.
(2000)
J. Brem et al.
Rhodanine hydrolysis leads to potent thioenolate mediated metallo-β-lactamase inhibition
Nat. Chem.
(2014)
J. Brem et al.
Structural basis of metallo-β-lactamase inhibition by captopril stereoisomers
Antimicrob. Agents Chemother.
(2016)

A.T. Brünger

Free R value: a novel statistical quantity for assessing the accuracy of crystal structures

Nature

(1992)

V.B. Chen et al.

MolProbity: all-atom structure validation for macromolecular crystallography

Acta Crystallogr. Sect. D Biol. Crystallogr.

(2010)

C.K. Das et al.

Hydrolysis of cephalexin and meropenem by New Delhi metallo-β-lactamase: the substrate protonation mechanism is drug dependent

Phys. Chem. Chem. Phys.

(2017)

J. Davies et al.

Origins and evolution of antibiotic resistance

Microbiol. Mol. Biol. Rev.

(2010)

J.D. Docquier et al.

High-resolution crystal structure of the subclass B3 metallo-β-lactamase BJP-1: rational basis for substrate specificity and interaction with sulfonamides

Antimicrob. Agents Chemother.

(2010)

P. Emsley et al.

Features and development of coot

Acta Crystallogr. Sect. D Biol. Crystallogr.

(2010)

W.S. Faraci et al.

Elimination of a good leaving group from the 3’-position of a cephalosporin need not be concerted with β-lactam ring opening: TEM-2 β-lactamase-catalyzed hydrolysis of pyridine-2-azo-4’-(”,N’-dimethylaniline) cephalosporin (PADAC) and of cephaloridine

J. Am. Chem. Soc.

(1984)

H. Feng et al.

Structural and mechanistic insights into NDM-1 catalyzed hydrolysis of cephalosporins

J. Am. Chem. Soc.

(2014)

L.P. Freedman et al.

The economics of reproducibility in preclinical research

PLoS Biol.

(2015)

J.-M. Frère et al.

From « an enzyme able to destroy penicillin » to carbapenemases: 70 years of β-lactamase misbehavior

Curr. Drug Targets

(2016)

M. Goto et al.

Inhibition of the metallo-beta-lactamase produced from Serratia marcescens by thiol compounds

Biol. Pharm. Bull.

(1997)

M. Grabowski et al.

A public database of macromolecular diffraction experiments

Acta Crystallogr. Sect. D Struct. Biol.

(2016)

M. Jaskolski

On the propagation of errors

Acta Crystallogr. Sect. D Biol. Crystallogr.

(2013)

Y. Kim et al.

NDM-1, the ultimate promiscuous enzyme: substrate recognition and catalytic mechanism

FASEB J.

(2013)

D.T. King et al.

New Delhi Metallo-β-lactamase: structural insights into β-lactam recognition and inhibition

J. Am. Chem. Soc.

(2012)

G.J. Kleywegt et al.

The Uppsala electron-density server

Acta Crystallogr. Sect. D Biol. Crystallogr.

(2004)

Cited by (44)

Multidrug-resistant non-typhoidal Salmonella in seasoned chicken meat
2024, Food Control
The current public health worry revolves around infections stemming from the consumption of meat contaminated with Salmonella spp., which has been linked to the growing issue of antimicrobial resistance. We investigated the presence of non-typhoidal Salmonella (NTS) in the retail of spicy chicken meat and its antimicrobial susceptibility profile. Forty establishments (supermarkets and butcheries) in Mid-West Brazil were visited in February and July 2017, totaling 80 samples. Salmonella isolation, molecular serotyping and antimicrobial analyses were performed. Of the total establishments, 18 sold meat NTS-positive, 6 of which were contaminated in the two visits, resulting in 24 contaminated samples (30%). Of these, the prevalent serovars were Heidelberg (58%–14/24), Minnesota (21%–05/24), Muenchen (17%–04/24) and Mbandaka (4%–01/24), but none was positive for Typhimurium and Enteritidis. Still, 55.6% (10/18) of the establishments did not have a veterinary inspection seal, and 50% (09/18) sold products beyond their expiration date. Worse still, 87.5% (21/24) of NTS isolates were resistant to at least one antimicrobial agent, being 66.7% (16/24) multidrug-resistant. The bla gene was detected in 41.6% (10/24), confirming resistance by β-lactamase enzymes. These findings highlighted the great risk of foodborne illness due to the possibility of contaminated seasoned chicken meat and transmission of bla resistant genes through the food chain.
Interactions of hydrolyzed β-lactams with the L1 metallo-β-lactamase: Crystallography supports stereoselective binding of cephem/carbapenem products
2023, Journal of Biological Chemistry
L1 is a dizinc subclass B3 metallo-β-lactamase (MBL) that hydrolyzes most β-lactam antibiotics and is a key resistance determinant in the Gram-negative pathogen Stenotrophomonas maltophilia, an important cause of nosocomial infections in immunocompromised patients. L1 is not usefully inhibited by MBL inhibitors in clinical trials, underlying the need for further studies on L1 structure and mechanism. We describe kinetic studies and crystal structures of L1 in complex with hydrolyzed β-lactams from the penam (mecillinam), cephem (cefoxitin/cefmetazole), and carbapenem (tebipenem, doripenem, and panipenem) classes. Despite differences in their structures, all the β-lactam-derived products hydrogen bond to Tyr33, Ser221, and Ser225 and are stabilized by interactions with a conserved hydrophobic pocket. The carbapenem products were modeled as Δ¹-imines, with (2S)-stereochemistry. Their binding mode is determined by the presence of a 1β-methyl substituent: the Zn-bridging hydroxide either interacts with the C-6 hydroxyethyl group (1β-hydrogen-containing carbapenems) or is displaced by the C-6 carboxylate (1β-methyl-containing carbapenems). Unexpectedly, the mecillinam product is a rearranged N-formyl amide rather than penicilloic acid, with the N-formyl oxygen interacting with the Zn-bridging hydroxide. NMR studies imply mecillinam rearrangement can occur nonenzymatically in solution. Cephem-derived imine products are bound with (3R)-stereochemistry and retain their 3′ leaving groups, likely representing stable endpoints, rather than intermediates, in MBL-catalyzed hydrolysis. Our structures show preferential complex formation by carbapenem- and cephem-derived species protonated on the equivalent (β) faces and so identify interactions that stabilize diverse hydrolyzed antibiotics. These results may be exploited in developing antibiotics, and β-lactamase inhibitors, that form long-lasting complexes with dizinc MBLs.
Thiols as a privileged scaffold against metallo-β-lactamases
2023, Privileged Scaffolds in Drug Discovery
Gram-negative bacteria producing metallo-β-lactamases (MβLs), which hydrolyze almost all β-lactams, including penicillins, cephalosporins, and carbapenems, are important contributors to β-lactam antibiotic resistance. β-Lactam/β-lactamase inhibitor combination therapy is a validated route to overcome this resistance, but MβL inhibitors are unavailable in the clinic. Here, we present an overview of representative thiol-based molecules as privileged scaffolds with broad-spectrum inhibition against all three subclasses of MβLs and their common inhibition principles. Challenges faced by the development of thiol-based inhibitors are analyzed in the clinical environment. Promising future directions for developing this research field are suggested with the hope of boosting drug discovery targeting MβLs to overcome antibiotic resistance.
Design, Synthesis, and Biological Evaluation of New 1H-Imidazole-2-Carboxylic Acid Derivatives as Metallo-β-Lactamase Inhibitors
2022, Bioorganic and Medicinal Chemistry
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 IC₅₀ 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.
Detecting anomalies in X-ray diffraction images using convolutional neural networks
2021, Expert Systems with Applications
Citation Excerpt :
Nevertheless, flaws in diffraction patterns such as loop scattering, strong background radiation, ice rings, diffuse scattering, or other artifacts are fairly common problems in macromolecular crystallography, where the signal to noise ratio is typically weak. When left unhandled, such anomalies can often deteriorate the quality of electron density maps and lead to model misinterpretation (Wlodawer et al., 2018; Raczynska, Shabalin, Minor, Wlodawer, & Jaskolski, 2018). Although several tools for quality assessment have appeared in recent years, most of them focus on electron density maps and structural models (Willard et al., 2003; Urzhumtseva, Afonine, Adams, & Urzhumtsev, 2009; Kowiel et al., 2018; Porebski, Sroka, Zheng, Cooper, & Minor, 2018), whereas those that analyze diffraction images only detect ice rings (Thorn et al., 2017).
Our understanding of life is based upon the interpretation of macromolecular structures and their dynamics. Almost 90% of currently known macromolecular models originated from electron density maps constructed using X-ray diffraction images. Even though diffraction images are critical for structure determination, due to their vast amounts and noisy, non-intuitive nature, their quality is rarely inspected. In this paper, we use recent advances in machine learning to automatically detect seven types of anomalies in X-ray diffraction images. For this purpose, we utilize a novel X-ray beam center detection algorithm, propose three different image representations, and compare the predictive performance of general-purpose classifiers and deep convolutional neural networks (CNNs). In benchmark tests on a set of 6,311 X-ray diffraction images, the proposed CNN achieved between 87% and 99% accuracy depending on the type of anomaly. Experimental results show that the proposed anomaly detection system can be considered suitable for early detection of sub-optimal data collection conditions and malfunctions at X-ray experimental stations.
Spectroscopic and biochemical characterization of metallo-β-lactamase IMP-1 with dicarboxylic, sulfonyl, and thiol inhibitors
2021, Bioorganic and Medicinal Chemistry
Citation Excerpt :
The authors speculated that one potential explanation of the time-dependent inhibition is isomerization of the enzyme-thiol complex with the thiol compound bound in a monodentate fashion to a μ-S bridged species; thiols with anionic substituents were proposed to isomerize quickly while thiols with neutral substituents isomerized slowly.21 While this may occur for some thiol compounds and IMP-1, a recent crystal structure shows monodentate binding of tiopronin to NDM-1.38 Similar to results on other MBLs, UV-vis, equilibrium dialyses, and native MS show that captopril, thiorphan, and tiopronin clearly form ternary complexes with IMP-1, coordinating the metal centers similar as NDM-1 and VIM-2.9,10
In an effort to probe the biophysical mechanisms of inhibition for ten previously-reported inhibitors of metallo-β-lactamases (MBL) with MBL IMP-1, equilibrium dialysis, metal analyses coupled with atomic absorption spectroscopy (AAS), native state mass spectrometry (native MS), and ultraviolet-visible spectrophotometry (UV-VIS) were used. 6-(1H-tetrazol-5-yl) picolinic acid (1T5PA), ANT431, D/l-captopril, thiorphan, and tiopronin were shown to form IMP-1/Zn(II)/inhibitor ternary complexes, while dipicolinic acid (DPA) and 4-(3-aminophenyl)pyridine-2,6-dicarboxylic acid (3AP-DPA) stripped some metal from the active site of IMP but also formed ternary complexes. DPA and 3AP-DPA stripped less metal from IMP-1 than from VIM-2 but stripped more metal from IMP-1 than from NDM-1. In contrast to a previous report, pterostilbene does not appear to bind to IMP-1 under our conditions. These results, along with previous studies, demonstrate similar mechanisms of inhibition toward different MBLs for different MBL inhibitors.

View all citing articles on Scopus

^☆: Dedication: As a birthday tribute, this work is dedicated to Dr. Zbigniew Dauter, an untiring advocate of the highest quality in structural research.

¹: Equal contribution.

View full text

A close look onto structural models and primary ligands of metallo-β-lactamases☆

Abstract

Section snippets

Introduction – reproducibility crisis in biomedical research

Model validation methods

NDM-1 with tiopronin (PDB ID 5a5z)

The ripple effect of sub-optimal and incorrect structural models

Acknowledgments

Drug Resist. Update

J. Mol. Biol.

Bioorganic Med. Chem. Lett.

Structure

Methods Enzymol.

Structure

PHENIX: a comprehensive Python-based system for macromolecular structure solution

Acta Crystallogr. Sect. D Biol. Crystallogr.

The protein data bank

Nucleic Acids Res.

Rhodanine hydrolysis leads to potent thioenolate mediated metallo-β-lactamase inhibition

Nat. Chem.

Structural basis of metallo-β-lactamase inhibition by captopril stereoisomers

Antimicrob. Agents Chemother.

Free R value: a novel statistical quantity for assessing the accuracy of crystal structures

Nature

MolProbity: all-atom structure validation for macromolecular crystallography

Acta Crystallogr. Sect. D Biol. Crystallogr.

Hydrolysis of cephalexin and meropenem by New Delhi metallo-β-lactamase: the substrate protonation mechanism is drug dependent

Phys. Chem. Chem. Phys.

Origins and evolution of antibiotic resistance

Microbiol. Mol. Biol. Rev.

High-resolution crystal structure of the subclass B3 metallo-β-lactamase BJP-1: rational basis for substrate specificity and interaction with sulfonamides

Antimicrob. Agents Chemother.

Features and development of coot

Acta Crystallogr. Sect. D Biol. Crystallogr.

Elimination of a good leaving group from the 3’-position of a cephalosporin need not be concerted with β-lactam ring opening: TEM-2 β-lactamase-catalyzed hydrolysis of pyridine-2-azo-4’-(”,N’-dimethylaniline) cephalosporin (PADAC) and of cephaloridine

J. Am. Chem. Soc.

Structural and mechanistic insights into NDM-1 catalyzed hydrolysis of cephalosporins

J. Am. Chem. Soc.

The economics of reproducibility in preclinical research

PLoS Biol.

From « an enzyme able to destroy penicillin » to carbapenemases: 70 years of β-lactamase misbehavior

Curr. Drug Targets

Inhibition of the metallo-beta-lactamase produced from Serratia marcescens by thiol compounds

Biol. Pharm. Bull.

A public database of macromolecular diffraction experiments

Acta Crystallogr. Sect. D Struct. Biol.

On the propagation of errors

Acta Crystallogr. Sect. D Biol. Crystallogr.

NDM-1, the ultimate promiscuous enzyme: substrate recognition and catalytic mechanism

FASEB J.

New Delhi Metallo-β-lactamase: structural insights into β-lactam recognition and inhibition

J. Am. Chem. Soc.

The Uppsala electron-density server

Acta Crystallogr. Sect. D Biol. Crystallogr.