Inhibiting Mycobacterium abscessus Cell Wall Synthesis: Using a Novel Diazabicyclooctane beta-Lactamase Inhibitor To Augment beta-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 beta-lactam and beta-lactamase inhibitor combinations that can effectively disrupt cell wall synthesis. To improve existing therapeutic regimens to address serious Mob infections, we evaluated the ability of durlobactam (DUR), a novel diazobicyclooctane beta-lactamase inhibitor to restore in vitro susceptibilities in combination with beta-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 MK ranges of <= 0.06 to 1 mu g/mL and an MIC50/MIC90 of <= 0.06/ 025 mu g/mL, respectively. We propose a model by which this enhancement may occur, DUR potently inhibited the beta-lactamase Bla(Mab) with a relative Michaelis constant (K-i (app)) of 4 x 10(-3) +/- 0.8 x 10(-3) mu M and acylation rate (k(2)/K) of 1 x 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 beta-lactams in eradicating Mab. IMPORTANCE Durlobactam (DUR) is a potent inhibitor of Bla(Mab) and provides protection of amoxicillin and imipenem against hydrolysis. DUR has intrinsic activity and forms stable acyl-enzyme complexes with Ldt(Mab2) and Ldt(Mab4). The ability of DUR to protect amoxicillin and imipenem against Bla(Mab) and its intrinsic activity along with the dual beta-lactam target redundancy can explain the rationale behind the potent activity of this combination.

Automated summary

Mycobacterium abscessus (Mab) infections are a growing threat to health, but combining DUR with beta-lactams can restore drug susceptibility. The triple drug combinations of CXM-DUR-AMOX and IMI-DUR-AMOX were the most effective in enhancing susceptibility.