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.

Metallo-β-Lactamases: Structure, Function, Epidemiology, Treatment Options, and the Development Pipeline.

Infections caused by resistant bacteria are nowadays too common, and some pathogens have even become resistant to multiple types of antibiotics, in which case few or even no treatments are available. In recent years, the most successful strategy in anti-infective drug discovery for the treatment of such problematic infections is the combination therapy "antibiotic + inhibitor of resistance". These inhibitors allow the repurposing of antibiotics that have already proven to be safe and effective for clinical use. Three main types of compounds have been developed to block the principal bacterial resistance mechanisms: (i) β-lactamase inhibitors; (ii) outer membrane permeabilizers; (iii) efflux pump inhibitors. This Perspective is focused on β-lactamase inhibitors that disable the most prevalent cause of antibiotic resistance in Gram-negative bacteria, i.e., the deactivation of the most widely used antibiotics, β-lactams (penicillins, cephalosporines, carbapenems, and monobactams), by the production of β-lactamases. An overview of the most recently identified β-lactamase inhibitors and of combination therapy is provided. The article also covers the mechanism of action of the different types of β-lactamase enzymes as a basis for inhibitor design and target inactivation.

β-Lactamase Inhibitors To Restore the Efficacy of Antibiotics against Superbugs.

Klebsiella pneumoniae is a Gram-negative organism and a major public health threat. In this study, we used whole-genome sequences to characterize 32 carbapenem-resistant K. pneumoniae (CRKP) and two carbapenem-resistant K. quasipneumoniae (CRKQ). Antimicrobial resistance was assessed using disk diffusion and E-test, while virulence was assessed in silico. The capsule type was determined by sequencing the wzi gene. The plasmid diversity was assessed by PCR-based replicon typing to detect the plasmid incompatibility (Inc) groups. The genetic relatedness was determined by multilocus sequence typing, pan-genome, and recombination analysis. All of the isolates were resistant to ertapenem together with imipenem and/or meropenem. Phenotypic resistance was due to blaOXA-48, blaNDM-1, blaNDM-7, or the coupling of ESBLs and outer membrane porin modifications. This is the first comprehensive study reporting on the WGS of CRKP and the first detection of CRKQ in the region. The presence and dissemination of CRKP and CRKQ, with some additionally having characteristics of hypervirulent clones such as the hypermucoviscous phenotype and the capsular type K2, are particularly concerning. Additionally, mining the completely sequenced K. pneumoniae genomes revealed the key roles of mobile genetic elements in the spread of antibiotic resistance and in understanding the epidemiology of these clinically significant pathogens.

Molecular Characterization of Carbapenem Resistant Klebsiella pneumoniae and Klebsiella quasipneumoniae Isolated from Lebanon.

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.

Principles and current strategies targeting metallo-β-lactamase mediated antibacterial resistance.

The sarco/endoplasmic reticulum calcium ATPase (SERCA), which plays a key role in the maintenance of Ca2+ ion homeostasis, is an extensively studied enzyme, the inhibition of which has a considerab...

Sarco/Endoplasmic Reticulum Calcium ATPase Inhibitors: Beyond Anticancer Perspective.

The clinical effectiveness of carbapenem antibiotics such as meropenem is becoming increasingly compromised by the spread of both metallo-β-lactamase (MBL) and serine-β-lactamase (SBL) enzymes on mobile genetic elements, stimulating research to find new β-lactamase inhibitors to be used in conjunction with carbapenems and other β-lactam antibiotics. Herein, we describe our initial exploration of a novel chemical series of metallo-β-lactamase inhibitors, from concept to efficacy, in a survival model using an advanced tool compound (ANT431) in conjunction with meropenem.

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SAR Studies Leading to the Identification of a Novel Series of Metallo-β-lactamase Inhibitors for the Treatment of Carbapenem-Resistant Enterobacteriaceae Infections That Display Efficacy in an Animal Infection Model

We have identified a series of tetrahydrocarbazoles as novel P-type ATPase inhibitors. Using a set of rationally designed analogues, we have analyzed their structure-activity relationship using functional assays, crystallographic data and computational modeling. We found that tetrahydrocarbazoles inhibit adenosine triphosphate (ATP) hydrolysis of the fungal H+-ATPase, depolarize the fungal plasma membrane and exhibit broad-spectrum antifungal activity. Comparative inhibition studies indicate that many tetrahydrocarbazoles also inhibit the mammalian Ca2+-ATPase (SERCA) and Na+,K+-ATPase with an even higher potency than Pma1. We have located the binding site for this compound class by crystallographic structure determination of a SERCA-tetrahydrocarbazole complex to 3.0 A resolution, finding that the compound binds to a region above the ion inlet channel of the ATPase. A homology model of the Candida albicans H+-ATPase based on this crystal structure, indicates that the compounds could bind to the same pocket and identifies pocket extensions that could be exploited for selectivity enhancement. The results of this study will aid further optimization towards selective H+-ATPase inhibitors as a new class of antifungal agents.

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Thomas David Pallin

Papers

SAR Studies Leading to the Identification of a Novel Series of Metallo-β-lactamase Inhibitors for the Treatment of Carbapenem-Resistant Enterobacteriaceae Infections That Display Efficacy in an Animal Infection Model

Tetrahydrocarbazoles are a novel class of potent P-type ATPase inhibitors with antifungal activity.