Kinetics of Avibactam Inhibition against Class A, C, and D β-Lactamases

• DOI: 10.1074/jbc.m113.485979
• Publication Date: 2013-08-03T00:17:31Z
• Authors: David E. Ehmann; Haris Jahić; Philip L. Ross; Rong-Fang Gu; Jun Hu; Thomas F. Durand-...; Sushmita Lahiri; Jason Thresher; Stephania Livchak; Ning Gao; Tiffany Palmer; Grant K. Walkup; Stewart L. Fisher
• Keywords: 0301 basic medicine; Magnetic Resonance Spectroscopy; Time Factors; Hydrolysis; Microbial Sensitivity Tests; Mass Spectrometry; beta-Lactamases; Anti-Bacterial Agents; Kinetics; 03 medical and health sciences; Drug Resistance, Bacterial; Enterobacter cloacae; Pseudomonas aeruginosa; Escherichia coli; Enzyme Inhibitors; Azabicyclo Compounds; Plasmids

Avibactam is a non-β-lactam β-lactamase inhibitor with a spectrum of activity that includes β-lactamase enzymes of classes A, C, and selected D examples. In this work acylation and deacylation rates were measured against the clinically important enzymes CTX-M-15, KPC-2, Enterobacter cloacae AmpC, Pseudomonas aeruginosa AmpC, OXA-10, and OXA-48. The efficiency of acylation (k2/Ki) varied across the enzyme spectrum, from 1.1 × 10(1) m(-1)s(-1) for OXA-10 to 1.0 × 10(5) for CTX-M-15. Inhibition of OXA-10 was shown to follow the covalent reversible mechanism, and the acylated OXA-10 displayed the longest residence time for deacylation, with a half-life of greater than 5 days. Across multiple enzymes, acyl enzyme stability was assessed by mass spectrometry. These inhibited enzyme forms were stable to rearrangement or hydrolysis, with the exception of KPC-2. KPC-2 displayed a slow hydrolytic route that involved fragmentation of the acyl-avibactam complex. The identity of released degradation products was investigated, and a possible mechanism for the slow deacylation from KPC-2 is proposed.