Inhibiting Mycobacterium abscessus Cell Wall Synthesis: Using a Novel Diazabicyclooctane b-Lactamase Inhibitor To Augment b-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 b-lactam and b-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 b-lactamase inhibitor to restore in vitro susceptibilities in combination with b-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 mg/mL and an MIC₅₀/MIC₉₀ of #0.06/ 0.25 mg/mL, respectively. We propose a model by which this enhancement may occur, DUR potently inhibited the b-lactamase Bla_Mab with a relative Michaelis constant (K_{i app}) of 4 x 102³ 6 0.8 x 102³ mM and acylation rate (k₂/K) of 1 x 10⁷ M2¹ s²¹. 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 b-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 b-lactam target redundancy can explain the rationale behind the potent activity of this combination.