The reaction mechanism of metallo-beta-lactamases is tuned by the conformation of an active site mobile loop

Carbapenems are "last resort" β-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-β-lactamases (MβLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all β-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MβLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MβLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different β-lactams in all MβLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MβLs.Fil: Palacios, Antonela Rocio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Mojica, María F.. Case Western Reserve University; Estados UnidosFil: Giannini, Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Taracila, Magdalena A.. Case Western Reserve University; Estados Unidos. Louis Stokes Veterans Affairs Medical Center; Estados UnidosFil: Bethel, Christopher R.. Louis Stokes Veterans Affairs Medical Center; Estados UnidosFil: Alzari, Pedro M.. Institut Pasteur de Paris; FranciaFil: Otero, Lisandro Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Llarrull, Leticia Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Bonomo, Robert A.. Case Western Reserve University; Estados UnidosFil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Reclaiming The Efficacy of β-Lactam–β-Lactamase Inhibitor Combinations: Avibactam Restores The Susceptibility of CMY-2-Producing Escherichia Coli to Ceftazidime

CMY-2 is a plasmid-encoded Ambler class C cephalosporinase that is widely disseminated in Enterobacteriaceae and is responsible for expanded-spectrum cephalosporin resistance. As a result of resistance to both ceftazidime and β-lactamase inhibitors in strains carrying blaCMY, novel β-lactam–β-lactamase inhibitor combinations are sought to combat this significant threat to β-lactam therapy. Avibactam is a bridged diazabicyclo [3.2.1]octanone non-β-lactam β-lactamase inhibitor in clinical development that reversibly inactivates serine β-lactamases. To define the spectrum of activity of ceftazidime-avibactam, we tested the susceptibilities of Escherichia coli clinical isolates that carry blaCMY-2 or blaCMY-69 and investigated the inactivation kinetics of CMY-2. Our analysis showed that CMY-2-containing clinical isolates of E. coli were highly susceptible to ceftazidime-avibactam (MIC90, ≤0.5 mg/liter); in comparison, ceftazidime had a MIC90 of \u3e128 mg/liter. More importantly, avibactam was an extremely potent inhibitor of CMY-2 β-lactamase, as demonstrated by a second-order onset of acylation rate constant (k2/K) of (4.9 ± 0.5) × 104 M−1 s−1 and the off-rate constant (koff) of (3.7 ± 0.4) ×10−4 s−1. Analysis of the reaction of avibactam with CMY-2 using mass spectrometry to capture reaction intermediates revealed that the CMY-2–avibactam acyl-enzyme complex was stable for as long as 24 h. Molecular modeling studies raise the hypothesis that a series of successive hydrogen-bonding interactions occur as avibactam proceeds through the reaction coordinate with CMY-2 (e.g., T316, G317, S318, T319, S343, N346, and R349). Our findings support the microbiological and biochemical efficacy of ceftazidime-avibactam against E. coli containing plasmid-borne CMY-2 and CMY-69

Reclaiming The Efficacy of β-Lactam–β-Lactamase Inhibitor Combinations: Avibactam Restores The Susceptibility of CMY-2-Producing Escherichia Coli to Ceftazidime

CMY-2 is a plasmid-encoded Ambler class C cephalosporinase that is widely disseminated in Enterobacteriaceae and is responsible for expanded-spectrum cephalosporin resistance. As a result of resistance to both ceftazidime and β-lactamase inhibitors in strains carrying blaCMY, novel β-lactam–β-lactamase inhibitor combinations are sought to combat this significant threat to β-lactam therapy. Avibactam is a bridged diazabicyclo [3.2.1]octanone non-β-lactam β-lactamase inhibitor in clinical development that reversibly inactivates serine β-lactamases. To define the spectrum of activity of ceftazidime-avibactam, we tested the susceptibilities of Escherichia coli clinical isolates that carry blaCMY-2 or blaCMY-69 and investigated the inactivation kinetics of CMY-2. Our analysis showed that CMY-2-containing clinical isolates of E. coli were highly susceptible to ceftazidime-avibactam (MIC90, ≤0.5 mg/liter); in comparison, ceftazidime had a MIC90 of \u3e128 mg/liter. More importantly, avibactam was an extremely potent inhibitor of CMY-2 β-lactamase, as demonstrated by a second-order onset of acylation rate constant (k2/K) of (4.9 ± 0.5) × 104 M−1 s−1 and the off-rate constant (koff) of (3.7 ± 0.4) ×10−4 s−1. Analysis of the reaction of avibactam with CMY-2 using mass spectrometry to capture reaction intermediates revealed that the CMY-2–avibactam acyl-enzyme complex was stable for as long as 24 h. Molecular modeling studies raise the hypothesis that a series of successive hydrogen-bonding interactions occur as avibactam proceeds through the reaction coordinate with CMY-2 (e.g., T316, G317, S318, T319, S343, N346, and R349). Our findings support the microbiological and biochemical efficacy of ceftazidime-avibactam against E. coli containing plasmid-borne CMY-2 and CMY-69

A Kinetic Analysis of The Inhibition of FOX-4 β-Lactamase, A Plasmid-Mediated AmpC Cephalosporinase, By Monocyclic β-lactams and Carbapenems

Abstract: Objectives: Class C β-lactamases are prevalent among Enterobacteriaceae; however, these enzymes are resistant to inactivation by commercially available β-lactamase inhibitors. In order to find novel scaffolds to inhibit class C β-lactamases, the comparative efficacy of monocyclic β-lactam antibiotics (aztreonam and the siderophore monosulfactam BAL30072), the bridged monobactam β-lactamase inhibitor BAL29880, and carbapenems (imipenem, meropenem, doripenem and ertapenem) were tested in kinetic assays against FOX-4, a plasmid-mediated class C β-lactamase (pmAmpC). Methods: The FOX-4 β-lactamase was purified. Steady-state kinetics, electrospray ionization mass spectrometry (ESI-MS) and ultraviolet difference (UVD) spectroscopy were conducted using the β-lactam scaffolds described. Results: The Ki values for the monocyclic β-lactams against FOX-4 β-lactamase were 0.04 ± 0.01 μM (aztreonam) and 0.66 ± 0.03 μM (BAL30072), and the Ki value for the bridged monobactam BAL29880 was 8.9 ± 0.5 μM. For carbapenems, the Ki values ranged from 0.27 ± 0.05 μM (ertapenem) to 2.3 ± 0.3 μM (imipenem). ESI-MS demonstrated the formation of stable covalent adducts when the monocyclic β-lactams and carbapenems were reacted with FOX-4 β-lactamase. UVD spectroscopy suggested the appearance of different chromophoric intermediates. Conclusions: Monocyclic β-lactam and carbapenem antibiotics are effective mechanism-based inhibitors of FOX-4 β-lactamase, a clinically important pmAmpC, and provide stimulus for the development of new inhibitors to inactivate plasmidic and chromosomal class C β-lactamases

Boronic Acid Transition State Inhibitors as Potent Inactivators of KPC and CTX-M β-Lactamases: Biochemical and Structural Analyses

Design of novel beta-lactamase inhibitors (BLIs) is one of the currently accepted strategies to combat the threat of cephalosporin and carbapenem resistance in Gram-negative bacteria. Boronic acid transition state inhibitors (BATSIs) are competitive, reversible BLIs that offer promise as novel therapeutic agents. In this study, the activities of two alpha-amido-beta-triazolylethaneboronic acid transition state inhibitors (S02030 and MB_076) targeting representative KPC (KPC-2) and CTX-M (CTX-M-96, a CTX-M-15-type extended-spectrum beta-lactamase [ESBL]) beta-lactamases were evaluated. The 50% inhibitory concentrations (IC(50)s) for both inhibitors were measured in the nanomolar range (2 to 135 nM). For S02030, the k(2)/K for CTX-M-96 (24,000 M-1 s(-1)) was twice the reported value for KPC-2 (12,000 M-1 s(-1)); for MB_076, the k(2)/K values ranged from 1,200 M-1 s(-1) (KPC-2) to 3,900 M-1 s(-1) (CTX-M-96). Crystal structures of KPC-2 with MB_076 (1.38-& ANGS; resolution) and S02030 and the in silico models of CTX-M-96 with these two BATSIs show that interaction in the CTX-M-96-S02030 and CTX-M-96-MB_076 complexes were overall equivalent to that observed for the crystallographic structure of KPC-2-S02030 and KPC-2-MB_076. The tetrahedral interaction surrounding the boron atom from S02030 and MB_076 creates a favorable hydrogen bonding network with S70, S130, N132, N170, and S237. However, the changes from W105 in KPC-2 to Y105 in CTX-M-96 and the missing residue R220 in CTX-M-96 alter the arrangement of the inhibitors in the active site of CTX-M-96, partially explaining the difference in kinetic parameters. The novel BATSI scaffolds studied here advance our understanding of structure-activity relationships (SARs) and illustrate the importance of new approaches to beta-lactamase inhibitor design

Click Chemistry in Lead Optimization of Boronic Acids as β-Lactamase Inhibitors

Boronic acid transition-state inhibitors (BATSIs) represent one of the most promising classes of β-lactamase inhibitors. Here we describe a new class of BATSIs, namely, 1-amido-2-triazolylethaneboronic acids, which were synthesized by combining the asymmetric homologation of boronates with copper-catalyzed azide-alkyne cycloaddition for the stereoselective insertion of the amido group and the regioselective formation of the 1,4-disubstituted triazole, respectively. This synthetic pathway, which avoids intermediate purifications, proved to be flexible and efficient, affording in good yields a panel of 14 BATSIs bearing three different R1 amide side chains (acetamido, benzylamido, and 2-thienylacetamido) and several R substituents on the triazole. This small library was tested against two clinically relevant class C β-lactamases from Enterobacter spp. and Pseudomonas aeruginosa. The Ki value of the best compound (13a) was as low as 4 nM with significant reduction of bacterial resistance to the combination of cefotaxime/13a

Extended-Spectrum AmpC Cephalosporinase in Acinetobacter baumannii: ADC-56 Confers Resistance to Cefepime▿

ADC-56, a novel extended-spectrum AmpC (ESAC) β-lactamase, was identified in an Acinetobacter baumannii clinical isolate. ADC-56 possessed an R148Q change compared with its putative progenitor, ADC-30, which enabled it to hydrolyze cefepime. Molecular modeling suggested that R148 interacted with Q267, E272, and I291 through a hydrogen bond network which constrained the H-10 helix. This permitted cefepime to undergo conformational changes in the active site, with the carboxyl interacting with R340, likely allowing for better binding and turnover

X-ray crystal structure of the extended-spectrum class C ²-lactamase, ADC-7, in apo form and in complex with a boronic acid transition state analog

Resistance to ²-lactam antibiotics in the pathogenic bacteria, Acinetobacter baumannii, presents one of the greatest challenges to contemporary antimicrobial chemotherapy. Much of this resistance derives from the expression of class C ²-lactamase enzymes, known as Acinetobacter-Derived Cephalosporinases (ADCs). In the search for novel therapies for multidrug resistant pathogens, determining the molecular structure of the enzyme active site is an important contribution. Here, we have determined the X-ray crystal structure of the extended-spectrum class C ²-lactamase, ADC-7, at 1.7 Å (P1 space group). In addition, while current ²-lactamase inhibitors are structurally similar to ²-lactam substrates and are not effective inactivators of the class C enzymes, boronic acid transition state inhibitors (BATSIs; chemically dissimilar to ²-lactams) were explored to inhibit ADC enzymes. In this study, we used a chiral, carboxy-aryl cephalothin analog BATSI, and the ADC-7 complex was determined at 2.0 Å resolution (P21 space group). In the complexed enzyme,the boronic acid makes several canonical interactions. The O1 oxygen is bound in the oxyanion hole, and the R1 amide group makes interactions with conserved residues Asn152 and Gln120. The carboxylate group of the inhibitor mimics the C4\u27 carboxylate found in ²-lactams. Asn289, Thr316, and Asn346 commonly comprise the C4\u27 carboxylate recognition residues in class C enzymes. However, in ADC-7, Asn289 is replaced with Glu289 and is pointed out of the active site. Interestingly, in ADC-7 complex, the inhibitor carboxylate group is observed to interact with Arg340, a residue that distinguishes ADC-7 from the related class C enzyme, AmpC. The ADC-7/boronic acid complex provides insight into recognition of non-²-lactam inhibitors by ADC enzymes and offers a novel starting point for the structure-based optimization of this class of novel ²-lactamase inhibitors against a clinically relevant resistance target

Click Chemistry in Lead Optimization of Boronic Acids as β‑Lactamase Inhibitors

Boronic acid transition-state inhibitors (BATSIs) represent one of the most promising classes of β-lactamase inhibitors. Here we describe a new class of BATSIs, namely, 1-amido-2-triazolylethaneboronic acids, which were synthesized by combining the asymmetric homologation of boronates with copper-catalyzed azide–alkyne cycloaddition for the stereoselective insertion of the amido group and the regioselective formation of the 1,4-disubstituted triazole, respectively. This synthetic pathway, which avoids intermediate purifications, proved to be flexible and efficient, affording in good yields a panel of 14 BATSIs bearing three different R1 amide side chains (acetamido, benzylamido, and 2-thienylacetamido) and several R substituents on the triazole. This small library was tested against two clinically relevant class C β-lactamases from Enterobacter spp. and Pseudomonas aeruginosa. The <i>K</i>_i value of the best compound (<b>13a</b>) was as low as 4 nM with significant reduction of bacterial resistance to the combination of cefotaxime/<b>13a</b>