Inhibiting Mycobacterium abscessus Cell Wall Synthesis: Using a Novel Diazabicyclooctane β-Lactamase Inhibitor To Augment β-Lactam Action

Mycobacterium abscessus (Mab) infections are a growing menace to the health of many patients, especially those suffering from structural lung disease and cystic fibrosis. With multidrug resistance a common feature and a growing understanding of peptidoglycan synthesis in Mab, it is advantageous to identify potent β-lactam and β-lactamase inhibitor combinations that can effectively disrupt cell wall synthesis. To improve existing therapeutic regimens to address serious Mab infections, we evaluated the ability of durlobactam (DUR), a novel diazobicyclooctane β-lactamase inhibitor to restore in vitro susceptibilities in combination with β-lactams and provide a biochemical rationale for the activity of this compound. In cell-based assays, susceptibility of Mab subsp. abscessus isolates to amoxicillin (AMOX), imipenem (IMI), and cefuroxime (CXM) was significantly enhanced with the addition of DUR. The triple drug combinations of CXM-DUR-AMOX and IMI-DUR-AMOX were most potent, with MIC ranges of ≤0.06 to 1 μg/mL and an MIC(50)/MIC(90) of ≤0.06/0.25 μg/mL, respectively. We propose a model by which this enhancement may occur, DUR potently inhibited the β-lactamase Bla(Mab) with a relative Michaelis constant (K(i) (app)) of 4 × 10(−3) ± 0.8 × 10(−3 )μM and acylation rate (k(2)/K) of 1 × 10(7) M(−1) s(−1). Timed mass spectrometry captured stable formation of carbamoyl-enzyme complexes between DUR and Ldt(Mab2-4) and Mab d,d-carboxypeptidase, potentially contributing to the intrinsic activity of DUR. Molecular modeling showed unique and favorable interactions of DUR as a Bla(Mab) inhibitor. Similarly, modeling showed how DUR might form stable Michaelis-Menten complexes with Ldt(Mab2-4) and Mab d,d-carboxypeptidase. The ability of DUR combined with amoxicillin or cefuroxime and imipenem to inactivate multiple targets such as d,d-carboxypeptidase and Ldt(Mab2,4) supports new therapeutic approaches using β-lactams in eradicating Mab.