ZN148 Is a Modular Synthetic Metallo-beta-Lactamase Inhibitor That Reverses Carbapenem Resistance in Gram-Negative Pathogens In Vivo

Abstract

Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (β-lactamases able to inactivate carbapenems) have been identified in both serine β-lactamase (SBL) and metallo-β-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited. Here, we present ZN148, a zinc-chelating MBL-inhibitor capable of restoring the bactericidal effect of meropenem and <i>in vitro</i> clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical <i>Enterobacterales</i> strains (<i>n</i> = 234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing <i>Klebsiella pneumoniae</i> in a murine neutropenic peritonitis model. ZN148 showed no inhibition of the human zinc-containing enzyme glyoxylase II at 500 μM, and no acute toxicity was observed in an <i>in vivo</i> mouse model with cumulative dosages up to 128 mg/kg. Biochemical analysis showed a time-dependent inhibition of MBLs by ZN148 and removal of zinc ions from the active site. Addition of exogenous zinc after ZN148 exposure only restored MBL activity by ∼30%, suggesting an irreversible mechanism of inhibition. Mass-spectrometry and molecular modeling indicated potential oxidation of the active site Cys221 residue. Overall, these results demonstrate the therapeutic potential of a ZN148-carbapenem combination against MBL-producing Gram-negative pathogens and that ZN148 is a highly promising MBL inhibitor that is capable of operating in a functional space not presently filled by any clinically approved compound.