Insights into the l,d-Transpeptidases and d,d-Carboxypeptidase of Mycobacterium abscessus: Ceftaroline, Imipenem, and Novel Diazabicyclooctane Inhibitors.

is a highly drug-resistant nontuberculous mycobacterium (NTM). Efforts to discover new treatments for infections are accelerating, with a focus on cell wall synthesis proteins ( l,d-transpeptidases 1 to 5 [Ldt to Ldt] and d,d-carboxypeptidase) that are targeted by β-lactam antibiotics. A challenge to this approach is the presence of chromosomally encoded β-lactamase (Bla). Using a mechanism-based approach, we found that a novel ceftaroline-imipenem combination effectively lowered the MICs of isolates (MIC ≤ 0.25 μg/ml; MIC ≤ 0.5 μg/ml). Combining ceftaroline and imipenem with a β-lactamase inhibitor, i.e., relebactam or avibactam, demonstrated only a modest effect on susceptibility compared to each of the β-lactams alone. In steady-state kinetic assays, Bla exhibited a lower (0.30 ± 0.03 μM for avibactam and 136 ± 14 μM for relebactam) and a higher acylation rate for avibactam (/= 3.4 × 10 ± 0.4 × 10 M s for avibactam and 6 × 10 ± 0.6 × 10 M s for relebactam). The / was nearly 10-fold lower for ceftaroline fosamil (0.007 ± 0.001 μM s) than for imipenem (0.056 ± 0.006 μM s). Timed mass spectrometry captured complexes of avibactam and Bla, Ldt, Ldt, Ldt, and d,d-carboxypeptidase, whereas relebactam bound only Bla, Ldt, and Ldt Interestingly, Ldt, Ldt, Ldt, Ldt, and d,d-carboxypeptidase bound only to imipenem when incubated with imipenem and ceftaroline fosamil. We next determined the binding constants of imipenem and ceftaroline fosamil for Ldt, Ldt, Ldt, and Ldt and showed that imipenem bound >100-fold more avidly than ceftaroline fosamil to Ldt and Ldt (e.g., or of Ldt = 0.01 ± 0.01 μM for imipenem versus 0.73 ± 0.08 μM for ceftaroline fosamil). Molecular modeling indicates that Ldt readily accommodates imipenem, but the active site must widen to ≥8 Å for ceftaroline to enter. Our analysis demonstrates that ceftaroline and imipenem binding to multiple targets (l,d-transpeptidases and d,d-carboxypeptidase) and provides a mechanistic rationale for the effectiveness of this dual β-lactam combination in infections.