NMR-filtered virtual screening leads to non-metal chelating metallo-β-lactamase inhibitors

Abstract

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.

Fig. 1. Outline mechanisms for (a) serine- (SBL) and (b) metallo-β-lactamase (MBL) catalysed hydrolysis. Note, in the case of the MBL variations of this mechanism are possible.

Fig. 2. ¹H CPMG NMR analyses reveal that compounds 16 and 17 bind to both di-Zn(ii) and apo-VIM-2 MBL. Binding studies of 16 (a) and 17 (b) to di-Zn(ii)-VIM-2 and apo-VIM-2 by ¹H CPMG NMR analyses. 16 and 17 bind to both di-Zn(ii) and apo-VIM-2 as indicated by signal intensity reduction in the presence of VIM-2. Assay mixtures contained 50 μM enzyme (either di-Zn(ii) VIM-2 in the presence of 50 μM Zn(ii) or apo-VIM-2), and 50 μM of the compound of interest buffered with 50 mM Tris-D₁₁, pH 7.5, in 90% H₂O and 10% D₂O. Black stars denote imidazole in the di-Zn(ii)-VIM-2 buffer (Fig. S15†). Note, the ¹H NMR spectra of 16 and 17 in DMSO-D₆ (Fig. S16†) are different from those in 50 mM Tris-D₁₁, pH 7.5, in 90% H₂O and 10% D₂O.

Fig. 3. Crystallographic analyses reveals compound 16 and 17 binding modes to VIM-2. (a) View from a crystal structure of VIM-2 in complex with compound 16 (PDB ID ; 5LE1) reveals that the inhibitor binds to form hydrophobic and electrostatic interactions with residues on the L3 and L10 loops (e.g., π–π stacking interactions with Phe61, and hydrogen-bonding interactions with Asn233; see Fig. S19 for further details of binding interaction). (b) View from a crystal structure of VIM-2 in complex with 17 (PDB ID ; 5LCA) reveals that 17 binds via a similar mode with that of 16. (c) Comparison of complex structures of VIM-2:16 and VIM-2:17 shows that both bind adjacent to zinc ions of VIM-2, but in a mode which does not involve direct zinc chelation. Distances between the oxygen atoms of the carboxylate of 16 and 17 and Zn2 are 4.63 Å and 5.57 Å, respectively. (d) The water molecule W3 is positioned to form tight hydrogen-bonding interactions with His196, Tyr224, and Asn233, and is important for binding the carboxylate of 16 and 17.

Fig. 4. Compounds 16 and 17 mimic interactions made by substrates. Selectivity profiles of 16 (a) and 17 (b) for class B MBLs and the class A serine β-lactamase TEM-1 (for which no inhibition was observed). (c) Comparison of VIM2 structures in complex with 16 (PDB ID ; 5LE1) and 17 (PDB ID ; 5LCA) with that of a representative substrate intermediate of a hydrolysed cephalosporin in complex with NDM-1 (PDB ID ; 4RL0) indicates that 16 and 17 have related binding modes to the cephalosporin substrate. Docking studies imply the binding modes of 16 and 17 may mimic the binding mode of the substrate prior to β-lactam hydrolysis (Fig. S22†).

Fig. 5. Crystallographic analyses reveals how the 3-oxoisoindoline-4-carboxylate derivatives bind to the VIM-2 MBL. (a) View from a crystal structure of VIM-2 in complex with 30 (PDB ID ; 5LCF). (b) Comparison of structures of VIM-2:17 and VIM-2:30 reveals the Asn233 side chain is unable to form hydrogen-bonds with the 3-oxoisoindoline-4-carboxylate of 17 due to its trifluoromethyl group, which may explain why 17 is less potent than 30 (Table 2). (c) View from a crystal structure of VIM-2 in complex with 35 (PDB ID ; 5LM6). (d) Comparison of VIM-2:30 and VIM-2:35 complex structures reveals 30 and 35 have the same binding mode. (e) View from a crystal structure of VIM-2 in complex with 42 (PDB ID ; 5LCH). (f) Comparison of structures of VIM-2:30 and VIM-2:42 reveals evidence for flexibility in the conformation of Tyr67 and Phe61 on the L3 loop, suggesting Tyr67 and Phe61 may play important roles in capturing substrates and delivering them to the zinc ions for hydrolysis.