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 in vitro clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical Enterobacterales strains (n = 234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing Klebsiella pneumoniae 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 in vivo 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.
Highlights
Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments
The main distinction between SBLs and MBLs is that SBLs possess an active site serine, whereas MBLs require the presence of zinc ions for activity. -Lactamases with carbapenemase activity have been identified in both of these families, including SBLs, such as KPC and OXA-48-like, and the MBLs NDM, VIM, and IMP [2]
Using ICP-MS for analysis of purified VIM-2 and NDM-1, we found that ZN148 removed ϳ1.8 and ϳ1.3 molar equivalents of zinc from VIM-2 and NDM-1, respectively (Fig. 6A)
Summary
Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Massspectrometry 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. The recent introduction of serine carbapenemase inhibitors such as avibactam, vaborbactam, and relebactam used in combination with -lactams has provided treatment options against serine carbapenemase-producing Gram-negative pathogens [7, 8] None of these -lactamase inhibitors possesses inhibitory activity against MBLs. The recent Italian outbreak of NDM-producing Enterobacteriaceae is significant due to its size and to the change in the epidemiology of carbapenem-resistant Enterobacteriaceae (CRE) from endemic KPC-producing CRE to NDM-producing CRE and the subsequent reduction in treatment options [9]. We report the preclinical development and characterization of a synthetic and modular MBL inhibitor (ZN148) with promising in vitro and in vivo efficacy
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