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.
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