title: LN-1-255, a penicillanic acid sulfone able to inhibit the class D carbapenemase OXA-48.
abstract: Carbapenemases are the most important mechanism responsible for carbapenem resistance in Enterobacteriaceae. Among carbapenemases, OXA-48 presents unique challenges as it is resistant to β-lactam inhibitors. Here, we test the capacity of the compound LN-1-255, a 6-alkylidene-2'-substituted penicillanic acid sulfone, to inhibit the activity of the carbapenemase OXA-48.
  
  
  
  The OXA-48 gene was cloned and expressed in Klebsiella pneumoniae and Escherichia coli in order to obtain MICs in the presence of inhibitors (clavulanic acid, tazobactam and sulbactam) and LN-1-255. OXA-48 was purified and steady-state kinetics was performed with LN-1-255 and tazobactam. The covalent binding mode of LN-1-255 with OXA-48 was studied by docking assays.
  
  
  
  Both OXA-48-producing clinical and transformant strains displayed increased susceptibility to carbapenem antibiotics in the presence of 4 mg/L LN-1-255 (2-32-fold increased susceptibility) and 16 mg/L LN-1-255 (4-64-fold increased susceptibility). Kinetic assays demonstrated that LN-1-255 is able to inhibit OXA-48 with an acylation efficiency (k2/K) of 10 ± 1 × 10(4) M(-1) s(-1) and a slow deacylation rate (koff) of 7 ± 1 × 10(-4) s(-1). IC50 was 3 nM for LN-1-255 and 1.5 μM for tazobactam. Lastly, kcat/kinact was 500-fold lower for LN-1-255 than for tazobactam.
  
  
  
  In these studies, carbapenem antibiotics used in combination with LN-1-255 are effective against the carbapenemase OXA-48, an important emerging mechanism of antibiotic resistance. This provides an incentive for further investigations to maximize the efficacy of penicillin sulfone inhibition of class D plasmid-carried Enterobacteriaceae carbapenemases.
authors.affiliation: Servicio de Microbiología-Instituto de Investigación Biomédica (INIBIC), 15006 A Coruña, Spain.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Vallejo
authors.affiliation: Servicio de Microbiología-Instituto de Investigación Biomédica (INIBIC), 15006 A Coruña, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Martínez-Guitián
authors.affiliation: Servicio de Microbiología-Instituto de Investigación Biomédica (INIBIC), 15006 A Coruña, Spain.
authors.forename: Juan C
authors.initials: JC
authors.lastname: Vázquez-Ucha
authors.affiliation: Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
authors.forename: Concepción
authors.initials: C
authors.lastname: González-Bello
authors.affiliation: Servicio de Microbiología-Instituto de Investigación Biomédica (INIBIC), 15006 A Coruña, Spain.
authors.forename: Margarita
authors.initials: M
authors.lastname: Poza
authors.affiliation: Department of Chemistry, Southern Methodist University, Dallas, TX 75275, USA.
authors.forename: John D
authors.initials: JD
authors.lastname: Buynak
authors.affiliation: Research Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, OH 44106, USA.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Bethel
authors.affiliation: Research Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, OH 44106, USA Departments of Biochemistry, Molecular Biology and Microbiology, Pharmacology and Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
authors.affiliation: Servicio de Microbiología-Instituto de Investigación Biomédica (INIBIC), 15006 A Coruña, Spain.
authors.forename: German
authors.initials: G
authors.lastname: Bou
authors.affiliation: Servicio de Microbiología-Instituto de Investigación Biomédica (INIBIC), 15006 A Coruña, Spain alejandro.beceiro.casas@sergas.es.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Beceiro
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015780:Carbapenems / Q000494:pharmacology; D003001:Cloning, Molecular; D003499:Cyclic S-Oxides / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D008826:Microbial Sensitivity Tests; D010406:Penicillins / Q000378:metabolism*; D011485:Protein Binding; D013407:Sulbactam / Q000378:metabolism*; D065093:beta-Lactamase Inhibitors / Q000378:metabolism*; D001618:beta-Lactamases / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D015780:Carbapenems; D003499:Cyclic S-Oxides; C538899:LN 1-255; D010406:Penicillins; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases; C108449:oxacillinase; D013407:Sulbactam