Repurposing of compound libraries yields new inhibitors of NDM-1 metallo-β-lactamase with diverse zinc-binding moieties

Antimicrobial resistance has emerged as a critical global public health threat, impacting human, animal and environmental health. An important mechanism of resistance is the production of β-lactamases, enzymes that hydrolyze the β-lactam ring, rendering β-lactam antibiotics ineffective. Metallo-β-lactamases (MBLs), which contain zinc ions in their active sites, are particularly challenging to counter as there are currently no inhibitors targeting these enzymes available on the market. Therefore, there is an urgent need for innovative drug discovery strategies to develop MBL-targeted therapies. New Delhi Metallo-β-Lactamase 1 (NDM-1) is the most widely disseminated MBL, with a global distribution in Enterobacterales . In this study, we used our library of fragment-sized chloroacetamides as a starting point to synthesize mercaptoacetamides as potential NDM-1 inhibitors. This resulted in a compound ( 14a ) with an IC 50 of 20 μM, which crystallography shows binds to NDM-1 in two different poses. Using this structure as a starting point for in silico design, we developed a series of larger thiol-based compounds designed to occupy more space in the active site and to utilize other novel zinc-binding groups. Although some showed minimal inhibition (which makes them valuable as decoys for metalloenzyme studies) one compound exhibited an IC 50 of 14 μM, with crystallography indicating that an additional aromatic group, compared to 14a , interacts with hydrophobic residues on an NDM-1 active site loop. These data identify promising scaffolds for the further development of potent MBL inhibitors and show the utility of repurposing chemical libraries to target clinically important enzymes. • Repurposing of a library of chloracetamides yielded new inhibitors of NDM-1 • Crystal structure of chloracetamide 14a in complex with NDM-1 was resolved • Zinc binding group library was constructed and used for in silico screening • An improved NDM-1 inhibitor 22b was found and evaluated structurally