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Resistance to β‐lactam antibiotics mediated by metallo‐β‐lactamases (MBLs) is a growing problem. We describe the use of protein‐observe 19F‐NMR (PrOF NMR) to study the dynamics of the São Paulo MBL (SPM‐1) from β‐lactam‐resistant Pseudomonas aeruginosa. Cysteinyl variants on the α3 and L3 regions, which flank the di‐ZnII active site, were selectively 19F‐labeled using 3‐bromo‐1,1,1‐trifluoroacetone. The PrOF NMR results reveal roles for the mobile α3 and L3 regions in the binding of both inhibitors and hydrolyzed β‐lactam products to SPM‐1. These results have implications for the mechanisms and inhibition of MBLs by β‐lactams and non‐β‐lactams and illustrate the utility of PrOF NMR for efficiently analyzing metal chelation, identifying new binding modes, and studying protein binding from a mixture of equilibrating isomers.

NMR-filtered virtual screening led to the identification of non-Zn(ii)-chelating metallo-β-lactamase inhibitors, which mimic interactions made by the bicyclic β-lactam antibiotic substrates as they initially bind to the enzymes., There are no clinically useful inhibitors of metallo-β-lactamases (MBLs), which are a growing problem because they hydrolyse almost all β-lactam antibacterials. Inhibition by most reported MBL inhibitors involves zinc ion chelation. A structure-based virtual screening approach combined with NMR filtering led to the identification of inhibitors of the clinically relevant Verona Integron-encoded MBL (VIM)-2. Crystallographic analyses reveal a new mode of MBL inhibition involving binding adjacent to the active site zinc ions, but which does not involve metal chelation. The results will aid efforts to develop new types of clinically useful inhibitors targeting MBLs/MBL-fold metallo-enzymes involved in antibacterial and anticancer drug resistance.