Structural and Kinetic Studies of the Potent Inhibition of Metallo-β-lactamases by 6-Phosphonomethylpyridine-2-carboxylates

Abstract

There are currently no clinically available inhibitors of metallo-β-lactamases (MBLs), enzymes that hydrolyze β-lactam antibiotics and confer resistance to Gram-negative bacteria. Here we present 6-phosphonomethylpyridine-2-carboxylates (PMPCs) as potent inhibitors of subclass B1 (IMP-1, VIM-2, and NDM-1) and B3 (L1) MBLs. Inhibition followed a competitive, slow-binding model without an isomerization step (IC50 values of 0.3–7.2 μM; Ki values of 0.03–1.5 μM). Minimum inhibitory concentration assays demonstrated potentiation of β-lactam (Meropenem) activity against MBL-producing bacteria, including clinical isolates, at concentrations at which eukaryotic cells remain viable. Crystal structures revealed unprecedented modes of binding of inhibitor to B1 (IMP-1) and B3 (L1) MBLs. In IMP-1, binding does not replace the nucleophilic hydroxide, and the PMPC carboxylate and pyridine nitrogen interact closely (2.3 and 2.7 Å, respectively) with the Zn2 ion of the binuclear metal site. The phosphonate group makes limited interactions but is 2.6 Å from the nucleophilic hydroxide. Furthermore, the presence of a water molecule interacting with the PMPC phosphonate and pyridine N–C2 π-bond, as well as the nucleophilic hydroxide, suggests that the PMPC binds to the MBL active site as its hydrate. Binding is markedly different in L1, with the phosphonate displacing both Zn2, forming a monozinc enzyme, and the nucleophilic hydroxide, while also making multiple interactions with the protein main chain and Zn1. The carboxylate and pyridine nitrogen interact with Ser221 and -223, respectively (3 Å distance). The potency, low toxicity, cellular activity, and amenability to further modification of PMPCs indicate these and similar phosphonate compounds can be further considered for future MBL inhibitor development.

Structural and biochemical analysis of the metallo‐β‐lactamase L1 from emerging pathogen Stenotrophomonas maltophilia revealed the subtle but distinct di‐metal scaffold for catalytic activity

“…L1 is the only B class β‐lactamase (BBL) that forms a tetramer and the formation of a tetramer is critical for the catalytic activity and/or substrate profile of L1 as shown by mutational study . All L1 MBL high‐resolution structures in this study, including the native form (L1‐Native), ligand‐bound forms, and ethylenediaminetetraacetic acid (EDTA)‐treated apo form (L1‐E) are virtually identical to those previously reported L1 structures with or without ligands bound . Root mean square deviations (RMSDs) on 264 or 265 Cα atoms are 0.3–0.4 Å, only a few residues deviate more than 1.0 Å (<2.0 Å) are at the surface of the protein away from the active site.…”

“…As shown in the reported structures, the active site is made of two Zn +2 (or Cd +2 or Cu +2 ) ions and the protein side chains coordinating metal ions at the bottom of the binding pocket. All the following atomic distances are observed in L1‐Native.…”

“…S. maltophilia L1 is a subclass B3 MBL, one of two chromosome‐encoded β‐lactamases (L2 belongs to a class A) . Several crystal structures of L1 and other B3 MBLs have been reported in the complexes with hydrolyzed antibiotics and inhibitors, which are supported by multiple biochemical analysis . B3 MBLs share a relatively low sequence identity of 23–35%; however, they are structurally well‐conserved particularly their di‐nuclear zinc active sites.…”

Investigation of Dipicolinic Acid Isosteres for the Inhibition of Metallo‐β‐Lactamases

“…Compounds 1-10 yield ar ange of chemical diversity (various acidic or metal-binding groups) ands how that the choice of the carboxylate isostere not only impactst he IC 50 value, but also the propensity for metal removal. [50] It is important to note that calculating K i values for metal-stripping inhibitors (such as 1 or 2)i sn ot appropriate because changes in IC 50 may not reflect changes in binding affinity.A vailable data suggestingp otentialv arying inhibition mechanismsb etween homologous MBLs and 2 (ternary complex formation with IMP-1, [49] but metal-stripping with NDM-1) was unexpected.T his difference may be due to a lower binding affinity for Zn II in the Zn 2 site of NDM-1 (K d = 2 mm) [45] relative to that of IMP-1 (K d = 0.3 mm), [51,52] resulting in more facile metal removal. Equilibrium dialysis data, in conjunction with UV/Vis spectroscopy of CoCoNDM-1, reveals differences in inhibition mechanism:i nhibition by 2 occurs primarily by Zn II removal, likely forming the inactive mono-Zn II NDM-1, whileinhibition by 4 is mainly via formation of at ernary complex at the dinuclearZ n II site.…”

“…As imple aryl ring (18 a), or an aryl ring with methoxy (18 b-d)o rh ydroxy substituent (18 e-g)w ereg enerally well-tolerated, with IC 50 = 0.20 AE 0.01-0.48 AE 0.01 mm,b ut did not lead to am arked decrease in IC 50 value compared with that of 2.G iven the structural similarity of 18 h-j (bearing the aniline substituent) to the previous reported inhibitor,( 4-(3-aminophenyl)pyridine-2,6-dicarboxylic acid) (Figure 1), no improvement in inhibition was observed relative to that of 2 (IC 50 = 0.24 AE 0.01-0.40 AE 0.02 mm). [49] Zn II ions are shownino range, coordinating ligandsa nd protein ribbona re showni n grey, 2 is shown in green,and ligand-protein interactions are shownw ith a yellow dashedline.T he image was rendered with Molecular Operating Environment (MOE). [49] Zn II ions are shownino range, coordinating ligandsa nd protein ribbona re showni n grey, 2 is shown in green,and ligand-protein interactions are shownw ith a yellow dashedline.T he image was rendered with Molecular Operating Environment (MOE).…”