ABSTRACT
The occurrence of resistances in Gram negative bacteria is steadily increasing to reach extremely worrying levels and one of the main causes of resistance is the massive spread of very efficient ß-lactamases which render most ß-lactam antibiotics useless. Herein, we report the development of a series of imino-analogues of ß-lactams (namely azetidinimines) as efficient non-covalent inhibitors of ß-lactamases. Despite the structural and mechanistic differences between serine-ß-lactamases KPC-2 and OXA-48 and metallo-ß-lactamase NDM-1, all three enzymes can be inhibited at a submicromolar level by compound 7dfm, which can also repotentiate imipenem against a resistant strain of Escherichia coli expressing NDM-1. We show that 7dfm can efficiently inhibit not only the three main clinically-relevant carbapenemases of Ambler classes A (KPC-2), B (NDM-1) and D (OXA-48) with Ki's below 0.3 µM, but also the cephalosporinase CMY-2 (class C, 86% inhibition at 10 µM). Our results pave the way for the development of a new structurally original family of non-covalent broad-spectrum inhibitors of ß-lactamases.
Subject(s)
Anti-Bacterial Agents/chemistry , Azetidines/chemistry , beta-Lactamase Inhibitors/chemistry , beta-Lactamases/chemistry , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Azetidines/metabolism , Binding Sites , Catalytic Domain , Cell Line , Cell Proliferation/drug effects , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gram-Negative Bacteria/drug effects , Humans , Inhibitory Concentration 50 , Microbial Sensitivity Tests , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Molecular Docking Simulation , Structure-Activity Relationship , beta-Lactamase Inhibitors/metabolism , beta-Lactamase Inhibitors/pharmacology , beta-Lactamases/genetics , beta-Lactamases/metabolismABSTRACT
BACKGROUND: KPC-like carbapenemases have spread worldwide with more than 30 variants identified that differ by single or double amino-acid substitutions. OBJECTIVES: To describe the steady-state kinetic parameters of KPC-28, which differs from KPC-2 by a H274Y substitution and the deletion of two amino acids (Δ242-GT-243). METHODS: The blaKPC-2, blaKPC-3, blaKPC-14 and blaKPC-28 genes were cloned into a pTOPO vector for susceptibility testing or into pET41b for overexpression, purification and subsequent kinetic parameter (Km, kcat) determination. Molecular docking experiments were performed to explore the role of the amino-acid changes in the carbapenemase activity. RESULTS: Susceptibility testing revealed that Escherichia coli producing KPC-28 displayed MICs that were lower for carbapenems and higher for ceftazidime and ceftazidime/avibactam as compared with KPC-2. The catalytic efficiencies of KPC-28 and KPC-14 for imipenem were 700-fold and 200-fold lower, respectively, than those of KPC-2, suggesting that Δ242-GT-243 in KPC-28 and KPC-14 is responsible for reduced carbapenem hydrolysis. Similarly, the H274Y substitution resulted in KPC-28 in a 50-fold increase in ceftazidime hydrolysis that was strongly reversed by clavulanate. CONCLUSIONS: We have shown that KPC-28 lacks carbapenemase activity, has increased ceftazidime hydrolytic activity and is strongly inhibited by clavulanate. KPC-28-producing E. coli isolates display an avibactam-resistant ESBL profile, which may be wrongly identified by molecular and immunochromatographic assays as the presence of a carbapenemase. Accordingly, confirmation of carbapenem hydrolysis will be mandatory with assays based solely on blaKPC gene or gene product detection.
