Durlobactam, a Diazabicyclooctane β-Lactamase Inhibitor, Inhibits BlaC and Peptidoglycan Transpeptidases of Mycobacterium tuberculosis

Peptidoglycan synthesis is an underutilized drug target in Mycobacterium tuberculosis (Mtb). Diazabicyclooctanes (DBOs) are a class of broad-spectrum β-lactamase inhibitors that also inhibit certain peptidoglycan transpeptidases that are important in mycobacterial cell wall synthesis. We evaluated the DBO durlobactam as an inhibitor of BlaC, the Mtb β-lactamase, and multiple Mtb peptidoglycan transpeptidases (PonA1, Ldt_Mt1, Ldt_Mt2, Ldt_Mt3, and Ldt_Mt5). Timed electrospray ionization mass spectrometry (ESI-MS) captured acyl-enzyme complexes with BlaC and all transpeptidases except Ldt_Mt5. Inhibition kinetics demonstrated durlobactam was a potent and efficient DBO inhibitor of BlaC (K_I app 9.2 ± 0.9 μM, k₂/K 5600 ± 560 M^–1 s^–1) and similar to clavulanate (K_I app 3.3 ± 0.6 μM, k₂/K 8400 ± 840 M^–1 s^–1); however, durlobactam had a lower turnover number (t_n = k_cat/k_inact) than clavulanate (1 and 8, respectively). K_I app values with durlobactam and clavulanate were similar for peptidoglycan transpeptidases, but ESI-MS captured durlobactam complexes at more time points. Molecular docking and simulation demonstrated several productive interactions of durlobactam in the active sites of BlaC, PonA1, and Ldt_Mt2. Antibiotic susceptibility testing was conducted on 11 Mtb isolates with amoxicillin, ceftriaxone, meropenem, imipenem, clavulanate, and durlobactam. Durlobactam had a minimum inhibitory concentration (MIC) range of 0.5–16 μg/mL, similar to the ranges for meropenem (1–32 μg/mL) and imipenem (0.5–64 μg/mL). In β-lactam + durlobactam combinations (1:1 mass/volume), MICs were lowered 4- to 64-fold for all isolates except one with meropenem–durlobactam. This work supports further exploration of novel β-lactamase inhibitors that target BlaC and Mtb peptidoglycan transpeptidases.