The New Delhi Metallo-β-lactamase-1 Covalent Inhibitors Derived from Cephalexin Effectively Reverse Meropenem...

New Delhi metallo-β-lactamase-1 (NDM-1) hydrolyzes carbapenems and most β-lactam antibiotics, posing a significant threat to global health and underscoring the urgent need for effective inhibitors. To address this unmet need, we designed a series of covalent NDM-1 inhibitors by introducing a selenazolone warhead into the cephalexin scaffold. Among them, compound 16a demonstrated the most potent inhibitory activity, with an IC₅₀ value of 3.27 μM. The combination of 16a and meropenem (Mem) synergistically lowered the MIC of Mem 8-16-fold in NDM-1-positive isolates and potently suppressed NDM-1-positive bacterial growth in a mouse infection model. Mechanistically, 16a forms a stable Se-S bond with Cys208 at the NDM-1 active site through its selenazolone warhead, enabling prolonged target engagement and sustained enzyme inhibition. Collectively, these findings demonstrate that covalent inhibitors derived from the cephalexin scaffold represent a promising strategy for combating NDM-1-mediated resistance, offering valuable leads for targeting metallo-β-lactamases.