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Biomolecules Jul 2018The ability of cell penetrating peptides (CPPs) to deliver biologically relevant cargos into cells is becoming more important as targets in the intracellular space...
The ability of cell penetrating peptides (CPPs) to deliver biologically relevant cargos into cells is becoming more important as targets in the intracellular space continue to be explored. We have developed two assays based on CPP-dependent, intracellular delivery of TEM-1 β-lactamase enzyme, a functional biological molecule comparable in size to many protein therapeutics. The first assay focuses on the delivery of full-length β-lactamase to evaluate the internalization potential of a CPP sequence. The second assay uses a split-protein system where one component of β-lactamase is constitutively expressed in the cytoplasm of a stable cell line and the other component is delivered by a CPP. The delivery of a split β-lactamase component evaluates the cytosolic delivery capacity of a CPP. We demonstrate that these assays are rapid, flexible and have potential for use with any cell type and CPP sequence. Both assays are validated using canonical and novel CPPs, with limits of detection from <500 nM to 1 µM. Together, the β-lactamase assays provide compatible tools for functional characterization of CPP activity and the delivery of biological cargos into cells.
Topics: Animals; CHO Cells; Cell Line; Cell-Penetrating Peptides; Cricetulus; Cytosol; Drug Delivery Systems; High-Throughput Screening Assays; Humans; beta-Lactamases
PubMed: 29997382
DOI: 10.3390/biom8030051 -
Clinical Microbiology and Infection :... Jan 2008Strains of Enterobacteriaceae producing an extended spectrum beta-lactamase have become a concern in medical bacteriology as regards both antimicrobial treatment and... (Review)
Review
Strains of Enterobacteriaceae producing an extended spectrum beta-lactamase have become a concern in medical bacteriology as regards both antimicrobial treatment and infection control in hospitals. Extended-spectrum beta-lactamase (ESBL) detection tests should accurately discriminate between bacteria producing these enzymes and those with other mechanisms of resistance to beta-lactams, e.g., broad-spectrum beta-lactamases, inhibitor-resistant beta-lactamases and cephalosporinase overproduction. Several phenotypic detection tests, based on the synergy between a third-generation cephalosporin and clavulanate, have been designed: the double-disk synergy test (DDST), ESBL Etests, and the combination disk method. These tests often need to be refined in order for them to detect an ESBL in some bacterial strains, such as those that also overproduce a cephalosporinase. The sensitivity of the DDST can be improved by reducing the distance between the disks of cephalosporins and clavulanate. The use of cefepime, a fourth-generation cephalosporin that is less rapidly inactivated by cephalosporinase than by ESBL, improves the detection of synergy with clavulanate when there is simultaneous stable hyperproduction of a cephalosporinase; alternatively, the cephalosporinase can be inactivated by performing phenotypic tests on a cloxacillin-containing agar. Some beta-lactamases can hydrolyse both third-generation cephalosporins and carbapenems, such as the metallo-beta-lactamases, which are not inhibited by clavulanate, but rather by EDTA. The production of an ESBL masked by a metallo-beta-lactamase can be detected by means of double inhibition by EDTA and clavulanate. Since extended-spectrum Ambler class D oxacillinases are weakly inhibited by clavulanate and not inhibited by EDTA, their detection is difficult in the routine laboratory.
Topics: Anti-Bacterial Agents; Cephalosporins; Clavulanic Acid; Enterobacteriaceae; Humans; Microbial Sensitivity Tests; Phenotype; Sensitivity and Specificity; beta-Lactamases
PubMed: 18154532
DOI: 10.1111/j.1469-0691.2007.01846.x -
Proceedings of the National Academy of... Mar 2020Gram-negative bacteria expressing class A β-lactamases pose a serious health threat due to their ability to inactivate all β-lactam antibiotics. The acyl-enzyme...
Gram-negative bacteria expressing class A β-lactamases pose a serious health threat due to their ability to inactivate all β-lactam antibiotics. The acyl-enzyme intermediate is a central milestone in the hydrolysis reaction catalyzed by these enzymes. However, the protonation states of the catalytic residues in this complex have never been fully analyzed experimentally due to inherent difficulties. To help unravel the ambiguity surrounding class A β-lactamase catalysis, we have used ultrahigh-resolution X-ray crystallography and the recently approved β-lactamase inhibitor avibactam to trap the acyl-enzyme complex of class A β-lactamase CTX-M-14 at varying pHs. A 0.83-Å-resolution CTX-M-14 complex structure at pH 7.9 revealed a neutral state for both Lys73 and Glu166. Furthermore, the avibactam hydroxylamine--sulfonate group conformation varied according to pH, and this conformational switch appeared to correspond to a change in the Lys73 protonation state at low pH. In conjunction with computational analyses, our structures suggest that Lys73 has a perturbed acid dissociation constant (pK) compared with acyl-enzyme complexes with β-lactams, hindering its function to deprotonate Glu166 and the initiation of the deacylation reaction. Further NMR analysis demonstrated Lys73 pK to be ∼5.2 to 5.6. Together with previous ultrahigh-resolution crystal structures, these findings enable us to follow the proton transfer process of the entire acylation reaction and reveal the critical role of Lys73. They also shed light on the stability and reversibility of the avibactam carbamoyl acyl-enzyme complex, highlighting the effect of substrate functional groups in influencing the protonation states of catalytic residues and subsequently the progression of the reaction.
Topics: Acylation; Azabicyclo Compounds; Bacterial Proteins; Binding Sites; Catalysis; Crystallography, X-Ray; Escherichia coli; Hydrogen-Ion Concentration; Models, Molecular; Molecular Conformation; Protein Conformation; Protons; beta-Lactamase Inhibitors; beta-Lactamases
PubMed: 32123084
DOI: 10.1073/pnas.1922203117 -
The Journal of Biological Chemistry Aug 2021β-Lactamase inhibitory protein (BLIP) consists of a tandem repeat of αβ domains conjugated by an interdomain loop and can effectively bind and inactivate class A...
β-Lactamase inhibitory protein (BLIP) consists of a tandem repeat of αβ domains conjugated by an interdomain loop and can effectively bind and inactivate class A β-lactamases, which are responsible for resistance of bacteria to β-lactam antibiotics. The varied ability of BLIP to bind different β-lactamases and the structural determinants for significant enhancement of BLIP variants with a point mutation are poorly understood. Here, we investigated the conformational dynamics of BLIP upon binding to three clinically prevalent class A β-lactamases (TEM1, SHV1, and PC1) with dissociation constants between subnanomolar and micromolar. Hydrogen deuterium exchange mass spectrometry revealed that the flexibility of the interdomain region was significantly suppressed upon strong binding to TEM1, but was not significantly changed upon weak binding to SHV1 or PC1. E73M and K74G mutations in the interdomain region improved binding affinity toward SHV1 and PC1, respectively, showing significantly increased flexibility of the interdomain region compared to the wild-type and favorable conformational changes upon binding. In contrast, more rigidity of the interfacial loop 135-145 was observed in these BLIP mutants in both free and bound states. Consistently, molecular dynamics simulations of BLIP exhibited drastic changes in the flexibility of the loop 135-145 in all complexes. Our results indicated for the first time that higher flexibility of the interdomain linker, as well as more rigidity of the interfacial loop 135-145, could be desirable determinants for enhancing inhibition of BLIP to class A β-lactamases. Together, these findings provide unique insights into the design of enhanced inhibitors.
Topics: Amino Acid Sequence; Bacteria; Bacterial Proteins; Drug Resistance, Bacterial; Molecular Dynamics Simulation; Protein Binding; Protein Domains; Protein Structural Elements; beta-Lactamase Inhibitors; beta-Lactamases
PubMed: 34302811
DOI: 10.1016/j.jbc.2021.100980 -
Clinical Microbiology Reviews Apr 2005The ascendancy of metallo-beta-lactamases within the clinical sector, while not ubiquitous, has nonetheless been dramatic; some reports indicate that nearly 30% of... (Review)
Review
The ascendancy of metallo-beta-lactamases within the clinical sector, while not ubiquitous, has nonetheless been dramatic; some reports indicate that nearly 30% of imipenem-resistant Pseudomonas aeruginosa strains possess a metallo-beta-lactamase. Acquisition of a metallo-beta-lactamase gene will invariably mediate broad-spectrum beta-lactam resistance in P. aeruginosa, but the level of in vitro resistance in Acinetobacter spp. and Enterobacteriaceae is less dependable. Their clinical significance is further embellished by their ability to hydrolyze all beta-lactams and by the fact that there is currently no clinical inhibitor, nor is there likely to be for the foreseeable future. The genes encoding metallo-beta-lactamases are often procured by class 1 (sometimes class 3) integrons, which, in turn, are embedded in transposons, resulting in a highly transmissible genetic apparatus. Moreover, other gene cassettes within the integrons often confer resistance to aminoglycosides, precluding their use as an alternative treatment. Thus far, the metallo-beta-lactamases encoded on transferable genes include IMP, VIM, SPM, and GIM and have been reported from 28 countries. Their rapid dissemination is worrisome and necessitates the implementation of not just surveillance studies but also metallo-beta-lactamase inhibitor studies securing the longevity of important anti-infectives.
Topics: Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Humans; beta-Lactam Resistance; beta-Lactamases; beta-Lactams
PubMed: 15831827
DOI: 10.1128/CMR.18.2.306-325.2005 -
Bioorganic & Medicinal Chemistry Letters Aug 2019β-Lactams are the most successful antibacterials, yet their use is threatened by resistance, importantly as caused by β-lactamases. β-Lactamases fall into two...
β-Lactams are the most successful antibacterials, yet their use is threatened by resistance, importantly as caused by β-lactamases. β-Lactamases fall into two mechanistic groups: the serine β-lactamases that utilise a covalent acyl-enzyme mechanism and the metallo β-lactamases that utilise a zinc-bound water nucleophile. Achieving simultaneous inhibition of both β-lactamase classes remains a challenge in the field. Vaborbactam is a boronate-based inhibitor that reacts with serine-β-lactamases to form covalent complexes that mimic tetrahedral intermediates in catalysis. Vaborbactam has recently been approved for clinical use in combination with the carbapenem meropenem. Here we show that vaborbactam moderately inhibits metallo-β-lactamases from all 3 subclasses (B1, B2 and B3), with a potency of around 20-100 fold below that by which it inhibits its current clinical targets, the Class A serine β-lactamases. This result contrasts with recent investigations of bicyclic boronate inhibitors, which potently inhibit subclass B1 MBLs but which presently lack activity against B2 and B3 enzymes. These findings indicate that cyclic boronate scaffolds have the potential to inhibit the full range of β-lactamases and justify further work on the development of boronates as broad-spectrum β-lactamase inhibitors.
Topics: Anti-Bacterial Agents; Boronic Acids; Humans; beta-Lactamases
PubMed: 31171422
DOI: 10.1016/j.bmcl.2019.05.031 -
FEMS Microbiology Reviews Nov 2010TEM-1 β-lactamase is one of the most well-known antibiotic resistance determinants around. It confers resistance to penicillins and early cephalosporins and has shown... (Review)
Review
TEM-1 β-lactamase is one of the most well-known antibiotic resistance determinants around. It confers resistance to penicillins and early cephalosporins and has shown an astonishing functional plasticity in response to the introduction of novel drugs derived from these antibiotics. Since its discovery in the 1960s, over 170 variants of TEM-1 - with different amino acid sequences and often resistance phenotypes - have been isolated in hospitals and clinics worldwide. Next to this well-documented 'natural' evolution, the in vitro evolution of TEM-1 has been the focus of attention of many experimental studies. In this review, we compare the natural and laboratory evolution of TEM-1 in order to address the question to what extent the evolution of antibiotic resistance can be repeated, and hence might have been predicted, under laboratory conditions. We also use the comparison to gain an insight into the adaptive relevance of hitherto uncharacterized substitutions present in clinical isolates and to predict substitutions not yet observed in nature. Based on new structural insights, we review what is known about substitutions in TEM-1 that contribute to the extension of its resistance phenotype. Finally, we address the clinical relevance of TEM alleles during the past decade, which has been dominated by the emergence of another β-lactamase, CTX-M.
Topics: Anti-Bacterial Agents; Bacteria; Bacterial Infections; Drug Resistance, Bacterial; Evolution, Molecular; Humans; Molecular Conformation; Mutation; beta-Lactamases
PubMed: 20412308
DOI: 10.1111/j.1574-6976.2010.00222.x -
Clinical Microbiology Reviews Oct 1995beta-Lactamases are the commonest single cause of bacterial resistance to beta-lactam antibiotics. Numerous chromosomal and plasmid-mediated types are known and may be... (Review)
Review
beta-Lactamases are the commonest single cause of bacterial resistance to beta-lactam antibiotics. Numerous chromosomal and plasmid-mediated types are known and may be classified by their sequences or phenotypic properties. The ability of a beta-lactamase to cause resistance varies with its activity, quantity, and cellular location and, for gram-negative organisms, the permeability of the producer strain. beta-Lactamases sometimes cause obvious resistance to substrate drugs in routine tests; often, however, these enzymes reduce susceptibility without causing resistance at current, pharmacologically chosen breakpoints. This review considers the ability of the prevalent beta-lactamases to cause resistance to widely used beta-lactams, whether resistance is accurately reflected in routine tests, and the extent to which the antibiogram for an organism can be used to predict the type of beta-lactamase that it produces.
Topics: Enterobacteriaceae; Gram-Negative Bacteria; Gram-Positive Bacteria; beta-Lactam Resistance; beta-Lactamases
PubMed: 8665470
DOI: 10.1128/CMR.8.4.557 -
FEBS Letters Nov 2015Metallo-β-lactamases are the latest resistance mechanism of pathogenic and opportunistic bacteria against carbapenems, considered as last resort drugs. The worldwide... (Review)
Review
Metallo-β-lactamases are the latest resistance mechanism of pathogenic and opportunistic bacteria against carbapenems, considered as last resort drugs. The worldwide spread of genes coding for these enzymes, together with the lack of a clinically useful inhibitor, have raised a sign of alarm. Inhibitor design has been mostly impeded by the structural diversity of these enzymes. Here we provide a critical review of mechanistic studies of the three known subclasses of metallo-β-lactamases, analyzed at the light of structural and mutagenesis investigations. We propose that these enzymes present a modular structure in their active sites that can be dissected into two halves: one providing the attacking nucleophile, and the second one stabilizing a negatively charged reaction intermediate. These are common mechanistic elements in all metallo-β-lactamases. Nucleophile activation does not necessarily requires a Zn(II) ion, but a Zn(II) center is essential for stabilization of the anionic intermediate. Design of a common inhibitor could be therefore approached based in these convergent mechanistic features despite the structural differences.
Topics: Bacteria; Biocatalysis; Crystallography, X-Ray; Drug Resistance, Microbial; Humans; Hydrolysis; beta-Lactamases
PubMed: 26297824
DOI: 10.1016/j.febslet.2015.08.015 -
Biochimie Jan 2017The microbial resistance to antibiotics is a genuine global threat. Consequently, a search of new inhibitors remains of acute importance due to the increasing spread of...
The microbial resistance to antibiotics is a genuine global threat. Consequently, a search of new inhibitors remains of acute importance due to the increasing spread of multidrug resistance. Here we present a new type of non-β-lactam β-lactamase inhibitor PA-34 based on natural phenoxyaniline, identified using computer-assisted screening of scaffolds related to those of known low-affinity inhibitors. The compound displays reversible competitive inhibition of bacterial β-lactamase TEM-171, with a K of 88 μM. Using enzyme kinetics, infra-red spectroscopy, fluorescence quenching and computer docking, we propose that the inhibitor binds at the entrance to the enzyme active site. This is a novel inhibition mechanism compared to binding covalently to the catalytic serine in the active site or non-covalently to the allosteric site. The residues involved in binding the inhibitor are conserved among molecular class A β-lactamases. The identified compound and its proposed binding mode may have a potential for a regulation of the catalytic activity of a wide range of class A β-lactamases. We also hypothesise that the presented route for finding non-β-lactam compounds may be an effective and durable approach for combating bacterial antibiotic resistance.
Topics: Acylation; Aniline Compounds; Bacterial Proteins; Binding Sites; Catalytic Domain; Computer Simulation; Drug Discovery; Electrophoresis, Polyacrylamide Gel; Fluorescence; Kinetics; Molecular Docking Simulation; Molecular Structure; Spectroscopy, Fourier Transform Infrared; beta-Lactamase Inhibitors; beta-Lactamases
PubMed: 27771370
DOI: 10.1016/j.biochi.2016.10.011