Discovery and characterization of a covalent inhibitor of New Delhi metallo β-lactamase.

New Delhi metallo β-lactamase (NDM-1) grants bacteria resistance to a broad spectrum of β-lactam antibiotics, including the last resort carbapenems. The absence of a nucleophilic serine in the active site of NDM-1 renders it resistant to canonical β-lactamase inhibitors that are used to treat β-lactam resistant bacteria. Herein, we report two new covalent irreversible inhibitors that target two highly conserved nucleophilic residues in the active site of NDM-1. N-Acryloyl-N-(isoquinoline-1-yl)acrylamide (2) inhibits NDM-1 in a time- and concentration-dependent manner without showing saturation kinetics (kinact/KI value = 4.6 ± 0.1 M-1s-1). Site-directed mutagenesis and ultraviolet photodissociation (UVPD) mass spectrometry reveal modification of the highly conserved active-site lysine (Lys211). The absence of saturation kinetics and incomplete inactivation of substrate turnover prompted the development of N-(8-hydroxyquinolin-2-yl)acrylamide (6), which appends a different metal binding motif to the acrylamide electrophile. Compound 6 also inhibits NDM-1 in a time- and concentration-dependent manner and shows saturation kinetics (KI = 167 ± 40 μM, kinact = 0.12 ± 0.01 min-1, kinact/KI = 12.0 ± 3.0 M-1s-1). Site-directed mutagenesis and UVPD mass spectrometry reveal modification of the zinc-coordinating active-site cysteine (Cys208). These results suggest that acrylamide can be used as an electrophilic warhead for targeting nucleophilic residues in the active site of NDM-1. Moreover, these inhibitors are promising scaffolds from which to design a covalent inhibitor that can serve as a codrug to restore the efficacy of β-lactams when treating NDM-1 expressing bacteria.