title: Exploring the Landscape of Diazabicyclooctane (DBO) Inhibition: Avibactam Inactivation of PER-2 β-Lactamase.
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.,k2/K= 2 × 103± 0.1 × 103M-1s-1, wherek2is the rate constant for acylation (carbamylation) andKis 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.,k2/Krange of ≈103M-1s-1) and not class A β-lactamases (i.e.,k2/Krange, 104to 105M-1s-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 (k2/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 againstEnterobacteriaceaeexpressingblaPER-2Our 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.
authors.affiliation: Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Microbiología, Inmunología y Biotecnología, Cátedra de Microbiología, Buenos Aires, Argentina.
authors.forename: Melina
authors.initials: M
authors.lastname: Ruggiero
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA.|Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.|Center for Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, Ohio USA.|Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Krisztina M
authors.initials: KM
authors.lastname: Papp-Wallace
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA.|Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Magdalena A
authors.initials: MA
authors.lastname: Taracila
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA.|Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Maria F
authors.initials: MF
authors.lastname: Mojica
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Bethel
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA.|Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Susan D
authors.initials: SD
authors.lastname: Rudin
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA.
authors.forename: Elise T
authors.initials: ET
authors.lastname: Zeiser
authors.affiliation: Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Microbiología, Inmunología y Biotecnología, Cátedra de Microbiología, Buenos Aires, Argentina.|Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Gutkind
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA robert.bonomo@va.gov ppower@ffyb.uba.ar.|Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.|Center for Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, Ohio USA.|Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, USA.|Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA.|Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
authors.affiliation: Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Microbiología, Inmunología y Biotecnología, Cátedra de Microbiología, Buenos Aires, Argentina robert.bonomo@va.gov ppower@ffyb.uba.ar.|Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.
authors.forename: Pablo
authors.identifier: 0000-0002-7051-9954
authors.initials: P
authors.lastname: Power
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D053961:Azabicyclo Compounds / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D001618:beta-Lactamases / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D053961:Azabicyclo Compounds; C543519:avibactam; C509814:beta-lactamase PER-2, Enterobacteriaceae; D001618:beta-Lactamases
keywords: avibactam; beta-lactamases; beta-lactams