scholarly journals Ligand-Dependent Disorder of the Ω Loop Observed in Extended-Spectrum SHV-Type β-Lactamase

2011 ◽  
Vol 55 (5) ◽  
pp. 2303-2309 ◽  
Author(s):  
Jared M. Sampson ◽  
Wei Ke ◽  
Christopher R. Bethel ◽  
S. R. R. Pagadala ◽  
Michael D. Nottingham ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Gram Negative Bacteria ◽  
Structural Alterations ◽  
Critical Amino Acid ◽  
Class A ◽  
Extended Spectrum ◽  
Loop Flexibility ◽  
Bacteria Resistance ◽  
Lactamase Inhibitor

ABSTRACTAmong Gram-negative bacteria, resistance to β-lactams is mediated primarily by β-lactamases (EC 3.2.6.5), periplasmic enzymes that inactivate β-lactam antibiotics. Substitutions at critical amino acid positions in the class A β-lactamase families result in enzymes that can hydrolyze extended-spectrum cephalosporins, thus demonstrating an “extended-spectrum” β-lactamase (ESBL) phenotype. Using SHV ESBLs with substitutions in the Ω loop (R164H and R164S) as target enzymes to understand this enhanced biochemical capability and to serve as a basis for novel β-lactamase inhibitor development, we determined the spectra of activity and crystal structures of these variants. We also studied the inactivation of the R164H and R164S mutants with tazobactam and SA2-13, a unique β-lactamase inhibitor that undergoes a distinctive reaction chemistry in the active site. We noted that the reducedKivalues for the R164H and R164S mutants with SA2-13 are comparable to those with tazobactam (submicromolar). The apo enzyme crystal structures of the R164H and R164S SHV variants revealed an ordered Ω loop architecture that became disordered when SA2-13 was bound. Important structural alterations that result from the binding of SA2-13 explain the enhanced susceptibility of these ESBL enzymes to this inhibitor and highlight ligand-dependent Ω loop flexibility as a mechanism for accommodating and hydrolyzing β-lactam substrates.

scholarly journals Crystal Structures of KPC-2 β-Lactamase in Complex with 3-Nitrophenyl Boronic Acid and the Penam Sulfone PSR-3-226

2012 ◽  
Vol 56 (5) ◽  
pp. 2713-2718 ◽  
Author(s):  
Wei Ke ◽  
Christopher R. Bethel ◽  
Krisztina M. Papp-Wallace ◽  
Sundar Ram Reddy Pagadala ◽  
Micheal Nottingham ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Boronic Acid ◽  
Carbonyl Oxygen ◽  
Oxyanion Hole ◽  
Covalent Interaction ◽  
Class A ◽  
Inactivation Rate Constant ◽  
Reversible Covalent ◽  
Acid Compound

ABSTRACTClass A carbapenemases are a major threat to the potency of carbapenem antibiotics. A widespread carbapenemase, KPC-2, is not easily inhibited by β-lactamase inhibitors (i.e., clavulanic acid, sulbactam, and tazobactam). To explore different mechanisms of inhibition of KPC-2, we determined the crystal structures of KPC-2 with two β-lactamase inhibitors that follow different inactivation pathways and kinetics. The first complex is that of a small boronic acid compound, 3-nitrophenyl boronic acid (3-NPBA), bound to KPC-2 with 1.62-Å resolution. 3-NPBA demonstrated aKmvalue of 1.0 ± 0.1 μM (mean ± standard error) for KPC-2 and blocks the active site by making a reversible covalent interaction with the catalytic S70 residue. The two boron hydroxyl atoms of 3-NPBA are positioned in the oxyanion hole and the deacylation water pocket, respectively. In addition, the aromatic ring of 3-NPBA provides an edge-to-face interaction with W105 in the active site. The structure of KPC-2 with the penam sulfone PSR-3-226 was determined at 1.26-Å resolution. PSR-3-226 displayed aKmvalue of 3.8 ± 0.4 μM for KPC-2, and the inactivation rate constant (kinact) was 0.034 ± 0.003 s−1. When covalently bound to S70, PSR-3-226 forms atrans-enamine intermediate in the KPC-2 active site. The predominant active site interactions are generated via the carbonyl oxygen, which resides in the oxyanion hole, and the carboxyl moiety of PSR-3-226, which interacts with N132, N170, and E166. 3-NPBA and PSR-3-226 are the first β-lactamase inhibitors to be trapped as an acyl-enzyme complex with KPC-2. The structural and inhibitory insights gained here could aid in the design of potent KPC-2 inhibitors.

scholarly journals Exploring the Landscape of Diazabicyclooctane (DBO) Inhibition: Avibactam Inactivation of PER-2 β-Lactamase

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Melina Ruggiero ◽  
Krisztina M. Papp-Wallace ◽  
Magdalena A. Taracila ◽  
Maria F. Mojica ◽  
Christopher R. Bethel ◽  
...  
Keyword(s):  
Active Site ◽  
Therapeutic Option ◽  
Structural Features ◽  
Atomic Mass ◽  
Stable Complex ◽  
Water Molecules ◽  
Gram Negative Bacteria ◽  
Biophysical Properties ◽  
Content Type ◽  
Class A

ABSTRACT PER β-lactamases are an emerging family of extended-spectrum β-lactamases (ESBL) found in Gram-negative bacteria. PER β-lactamases are unique among class A enzymes as they possess an inverted omega (Ω) loop and extended B3 β-strand. These singular structural features are hypothesized to contribute to their hydrolytic profile against oxyimino-cephalosporins (e.g., cefotaxime and ceftazidime). Here, we tested the ability of avibactam (AVI), a novel non-β-lactam β-lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k 2/K = 2 × 103 ± 0.1 × 103 M−1 s−1, where k 2 is the rate constant for acylation (carbamylation) and K is the equilibrium constant) that were obtained when AVI was tested were reminiscent of values observed testing the inhibition by AVI of class C and D β-lactamases (i.e., k 2/K range of ≈103 M−1 s−1) and not class A β-lactamases (i.e., k 2/K range, 104 to 105 M−1 s−1). Once AVI was bound, a stable complex with PER-2 was observed via mass spectrometry (e.g., 31,389 ± 3 atomic mass units [amu] → 31,604 ± 3 amu for 24 h). Molecular modeling of PER-2 with AVI showed that the carbonyl of AVI was located in the oxyanion hole of the β-lactamase and that the sulfate of AVI formed interactions with the β-lactam carboxylate binding site of the PER-2 β-lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 β-strands) were observed and may affect the binding of necessary catalytic water molecules, thus slowing acylation (k 2/K) of AVI onto PER-2. Similar electrostatics and hydrophobicity of the active site were also observed between OXA-48 and PER-2, while CTX-M-15 was more hydrophilic. To demonstrate the ability of AVI to overcome the enhanced cephalosporinase activity of PER-2 β-lactamase, we tested different β-lactam–AVI combinations. By lowering MICs to ≤2 mg/liter, the ceftaroline-AVI combination could represent a favorable therapeutic option against Enterobacteriaceae expressing bla PER-2. Our studies define the inactivation of the PER-2 ESBL by AVI and suggest that the biophysical properties of the active site contribute to determining the efficiency of inactivation.

scholarly journals Microbiological characterization of VNRX-5236: a broad spectrum β-lactamase inhibitor for rescue of the orally bioavailable cephalosporin ceftibuten as a carbapenem-sparing agent against strains of Enterobacterales expressing extended spectrum β-lactamases and serine carbapenemases.

2021 ◽  
Author(s):  
Cassandra L. Chatwin ◽  
Jodie C. Hamrick ◽  
Robert E. L. Trout ◽  
Cullen L. Myers ◽  
Susan M. Cusick ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Clinical Isolates ◽  
Class A ◽  
Extended Spectrum ◽  
Oral Agents ◽  
Class C ◽  
Orally Bioavailable ◽  
Lactamase Inhibitor

There is an urgent need for oral agents to combat resistant gram-negative pathogens. Here we describe the characterization of VNRX-5236, a broad-spectrum boronic acid β-lactamase inhibitor (BLI) and its orally bioavailable etzadroxil prodrug, VNRX-7145. VNRX-7145 is being developed in combination with ceftibuten, an oral cephalosporin, to combat strains of Enterobacterales expressing extended spectrum β-lactamases (ESBLs) and serine carbapenemases. VNRX-5236 is a reversible covalent inhibitor of serine β-lactamases, with inactivation efficiencies on the order of 104 M−1. sec−1, and prolonged active site residence times (t1/2, 5 to 46 min). The spectrum of inhibition includes Ambler class A ESBLs, class C cephalosporinases, and class A and D carbapenemases (KPC and OXA-48, respectively). Rescue of ceftibuten by VNRX-5236 (fixed at 4 μg/mL) in isogenic strains of E. coli expressing class A, C or D β-lactamases demonstrated an expanded spectrum of activity relative to oral comparators including investigational penems, sulopenem and tebipenem. VNRX-5236 rescued ceftibuten activity in clinical isolates of Enterobacterales expressing ESBLs (MIC90 = 0.25 μg/mL), KPCs (MIC90 = 1 μg/mL), class C cephalosporinases (MIC90 = 1 μg/mL) and OXA-48-type carbapenemases (MIC90 = 1 μg/mL). Frequency of resistance studies demonstrated a low propensity for recovery of resistant variants at 4× the MIC of the ceftibuten/VNRX-5236 combination. In vivo, whereas ceftibuten alone was ineffective (ED50, >128 mg/kg), ceftibuten/VNRX-7145 administered orally protected mice from lethal septicemia caused by K. pneumoniae producing KPC carbapenemase (ED50, 12.9 mg/kg). The data demonstrate potent, broad-spectrum rescue of ceftibuten activity by VNRX-5236 in clinical isolates of cephalosporin-resistant and carbapenem-resistant Enterobacterales.

scholarly journals Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate

2016 ◽  
Vol 61 (1) ◽  
Author(s):  
Venu Gopal Vandavasi ◽  
Patricia S. Langan ◽  
Kevin L. Weiss ◽  
Jerry M. Parks ◽  
Jonathan B. Cooper ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Active Site Residues ◽  
Hydrogen Atoms ◽  
Content Type ◽  
X Ray Crystallography ◽  
General Base ◽  
Class A ◽  
Extended Spectrum ◽  
Enzyme Intermediate

ABSTRACT The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M β-lactamase but not for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A β-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, as previously proposed.

scholarly journals Probing the Mechanism of Inactivation of the FOX-4 Cephamycinase by Avibactam

2018 ◽  
Vol 62 (5) ◽  
pp. e02371-17 ◽  
Author(s):  
Michiyoshi Nukaga ◽  
Krisztina M. Papp-Wallace ◽  
Tyuji Hoshino ◽  
Scott T. Lefurgy ◽  
Christopher R. Bethel ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bonding ◽  
Single Amino Acid ◽  
Content Type ◽  
Class A ◽  
Hydrogen Bonding Interactions ◽  
Extended Spectrum ◽  
Inhibitor Combination ◽  
Lactamase Inhibitor ◽  
Inhibition Analysis

ABSTRACTCeftazidime-avibactam is a “second-generation” β-lactam–β-lactamase inhibitor combination that is effective againstEnterobacteriaceaeexpressing class A extended-spectrum β-lactamases, class A carbapenemases, and/or class C cephalosporinases. Knowledge of the interactions of avibactam, a diazabicyclooctane with different β-lactamases, is required to anticipate future resistance threats. FOX family β-lactamases possess unique hydrolytic properties with a broadened substrate profile to include cephamycins, partly as a result of an isoleucine at position 346, instead of the conserved asparagine found in most AmpCs. Interestingly, a single amino acid substitution at N346 in theCitrobacterAmpC is implicated in resistance to the aztreonam-avibactam combination. In order to understand how diverse active-site topologies affect avibactam inhibition, we tested a panel of clinicalEnterobacteriaceaeisolates producingblaFOXusing ceftazidime-avibactam, determined the biochemical parameters for inhibition using the FOX-4 variant, and probed the atomic structure of avibactam with FOX-4. Avibactam restored susceptibility to ceftazidime for most isolates producingblaFOX; two isolates, one expressingblaFOX-4and the other producingblaFOX-5, displayed an MIC of 16 μg/ml for the combination. FOX-4 possessed ak2/Kvalue of 1,800 ± 100 M−1· s−1and an off rate (koff) of 0.0013 ± 0.0003 s−1. Mass spectrometry showed that the FOX-4–avibactam complex did not undergo chemical modification for 24 h. Analysis of the crystal structure of FOX-4 with avibactam at a 1.5-Å resolution revealed a unique characteristic of this AmpC β-lactamase. Unlike in thePseudomonas-derived cephalosporinase 1 (PDC-1)–avibactam crystal structure, interactions (e.g., hydrogen bonding) between avibactam and position I346 in FOX-4 are not evident. Furthermore, another residue is not observed to be close enough to compensate for the loss of these critical hydrogen-bonding interactions. This observation supports findings from the inhibition analysis of FOX-4; FOX-4 possessed the highestKd(dissociation constant) value (1,600 nM) for avibactam compared to other AmpCs (7 to 660 nM). Medicinal chemists must consider the properties of extended-spectrum AmpCs, such as the FOX β-lactamases, for the design of future diazabicyclooctanes.

scholarly journals Multiple substitutions at position 104 of β-lactamase TEM-1: assessing the role of this residue in substrate specificity

1995 ◽  
Vol 305 (1) ◽  
pp. 33-40 ◽  
Author(s):  
A Petit ◽  
L Maveyraud ◽  
F Lenfant ◽  
J P Samama ◽  
R Labia ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Kinetic Properties ◽  
Third Generation ◽  
Wild Type Enzyme ◽  
Beta Lactamases ◽  
Class A ◽  
Extended Spectrum ◽  
Third Generation Cephalosporins

Residue 104 is frequently mutated from a glutamic acid to a lysine in the extended-spectrum TEM beta-lactamases responsible for the resistance to third-generation cephalosporins in clinical Gram negative strains. Among class A beta-lactamases, it is the most variable residue within a highly conserved loop which delineates one side of the active site of the enzymes. To investigate the role of this residue in the extended-spectrum phenotype, it has been replaced by serine, threonine, lysine, arginine, tyrosine and proline. All these substitutions yield active enzymes, with no drastic changes in kinetic properties compared with the wild-type enzyme, except with cefaclor, but an overall improved affinity for second- and third-generation cephalosporins. Only mutant E104K exhibits a significant ability to hydrolyse cefotaxime. Molecular modelling shows that the substitutions have generally no impact on the conformation of the 101-111 loop as the side chains of residues at position 104 are all turned towards the solvent. Unexpectedly, the E104P mutant turns out to be the most efficient enzyme. All our results argue in favour of an indirect role for this residue 104 in the substrate specificity of the class A beta-lactamases. This residue contributes to the precise positioning of residues 130-132 which are involved in substrate binding and catalysis. Changing residue 104 could also modify slightly the local electrostatic potential in this part of the active site. The limited kinetic impact of the mutations at this position have to be analysed in the context of the microbiological problem of resistance to third-generation cephalosporins. Although mutation E104K improves the ability of the enzyme to hydrolyse these compounds, it is not sufficient to confer true resistance, and is always found in clinical isolates associated with at least one mutation at another part of the active site. It is the combined effect of the two mutations that synergistically enhances the hydrolytic capability of the enzyme towards third-generation cephalosporins.

scholarly journals Insights Into the Inhibition of MOX-1 β-Lactamase by S02030, a Boronic Acid Transition State Inhibitor

2021 ◽  
Vol 12 ◽  
Author(s):  
Tatsuya Ishikawa ◽  
Nayuta Furukawa ◽  
Emilia Caselli ◽  
Fabio Prati ◽  
Magdalena A. Taracila ◽  
...  
Keyword(s):  
Transition State ◽  
Active Site ◽  
Boronic Acid ◽  
Laboratory Strain ◽  
Multidrug Resistant ◽  
Mechanisms Of Action ◽  
Gram Negative Bacteria ◽  
Class A ◽  
Fixed Concentration ◽  
Active Site Cavity

The rise of multidrug resistant (MDR) Gram-negative bacteria has accelerated the development of novel inhibitors of class A and C β-lactamases. Presently, the search for novel compounds with new mechanisms of action is a clinical and scientific priority. To this end, we determined the 2.13-Å resolution crystal structure of S02030, a boronic acid transition state inhibitor (BATSI), bound to MOX-1 β-lactamase, a plasmid-borne, expanded-spectrum AmpC β-lactamase (ESAC) and compared this to the previously reported aztreonam (ATM)-bound MOX-1 structure. Superposition of these two complexes shows that S02030 binds in the active-site cavity more deeply than ATM. In contrast, the SO3 interactions and the positional change of the β-strand amino acids from Lys315 to Asn320 were more prominent in the ATM-bound structure. MICs were performed using a fixed concentration of S02030 (4 μg/ml) as a proof of principle. Microbiological evaluation against a laboratory strain of Escherichia coli expressing MOX-1 revealed that MICs against ceftazidime are reduced from 2.0 to 0.12 μg/ml when S02030 is added at a concentration of 4 μg/ml. The IC50 and Ki of S02030 vs. MOX-1 were 1.25 ± 0.34 and 0.56 ± 0.03 μM, respectively. Monobactams such as ATM can serve as informative templates for design of mechanism-based inhibitors such as S02030 against ESAC β-lactamases.

scholarly journals Activity of NXL104 in Combination with β-Lactams against Genetically Characterized Escherichia coli and Klebsiella pneumoniae Isolates Producing Class A Extended-Spectrum β-Lactamases and Class C β-Lactamases

2011 ◽  
Vol 55 (5) ◽  
pp. 2434-2437 ◽  
Author(s):  
P. R. S. Lagacé-Wiens ◽  
F. Tailor ◽  
P. Simner ◽  
M. DeCorby ◽  
J. A. Karlowsky ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
The Novel ◽  
Content Type ◽  
E Coli ◽  
Class A ◽  
Extended Spectrum ◽  
Class C ◽  
Lactamase Inhibitor

ABSTRACTThe novel non-β-lactam β-lactamase inhibitor NXL104, in combination with cefepime, ceftazidime, ceftriaxone, amdinocillin, and meropenem, was tested against 190 extended-spectrum β-lactamase (ESBL)-producingEscherichia coliandKlebsiella pneumoniaeisolates, 94 AmpC-hyperproducingE. coliisolates, and 8 AmpC/ESBL-coexpressingE. coliisolates. NXL104 restored 100% susceptibility to the partner cephalosporins for all isolates tested. Amdinocillin and meropenem MICs were modestly improved (2 to 32 times lower) by NXL104. These results suggest that NXL104 may be useful in combination with β-lactams for the treatment of infections caused by ESBL- and AmpC-producingEnterobacteriaceae.

scholarly journals Crystal Structure of the Mycobacterium fortuitum Class A β-Lactamase: Structural Basis for Broad Substrate Specificity

2006 ◽  
Vol 50 (7) ◽  
pp. 2516-2521 ◽  
Author(s):  
Eric Sauvage ◽  
Eveline Fonzé ◽  
Birgit Quinting ◽  
Moreno Galleni ◽  
Jean-Marie Frère ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Broad Spectrum ◽  
Active Site ◽  
Bacterial Resistance ◽  
Specific Activity ◽  
Structural Basis ◽  
Mycobacterium Fortuitum ◽  
Bacterial Strains ◽  
Class A ◽  
Extended Spectrum

ABSTRACT β-Lactamases are the main cause of bacterial resistance to penicillins and cephalosporins. Class A β-lactamases, the largest group of β-lactamases, have been found in many bacterial strains, including mycobacteria, for which no β-lactamase structure has been previously reported. The crystal structure of the class A β-lactamase from Mycobacterium fortuitum (MFO) has been solved at 2.13-Å resolution. The enzyme is a chromosomally encoded broad-spectrum β-lactamase with low specific activity on cefotaxime. Specific features of the active site of the class A β-lactamase from M. fortuitum are consistent with its specificity profile. Arg278 and Ser237 favor cephalosporinase activity and could explain its broad substrate activity. The MFO active site presents similarities with the CTX-M type extended-spectrum β-lactamases but lacks a specific feature of these enzymes, the VNYN motif (residues 103 to 106), which confers on CTX-M-type extended-spectrum β-lactamases a more efficient cefotaximase activity.

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