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The rise of Gram-negative pathogens expressing metallo-β-lactamases (MBLs) is a growing concern, threatening the efficacy of β-lactam antibiotics, in particular, the carbapenems. There are no inhibitors of MBLs in current clinical use. Aspergillomarasmine A (AMA) is an MBL inhibitor isolated from Aspergillus versicolor with the ability to rescue meropenem activity in MBL-producing bacteria both in vitro and in vivo. Here, we systematically explored the pairing of AMA with six β-lactam antibiotic partners against 19 MBLs from three subclasses (B1, B2, and B3). Cell-based assays performed with Escherichia coli and Klebsiella pneumoniae showed that bacteria producing NDM-1 and VIM-2 of subclass B1 were the most susceptible to AMA inhibition, whereas bacteria producing CphA2 and AIM-1 of subclasses B2 and B3, respectively, were the least sensitive. Intracellular antibiotic accumulation assays and in vitro enzyme assays demonstrated that the efficacy of AMA/β-lactam combinations did not correlate with outer membrane permeability or drug efflux. We determined that the optimal β-lactam partners for AMA are the carbapenem antibiotics and that the efficacy of AMA is linked to the Zn2+ affinity of specific MBLs.

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The aminopolycarboxylic acid aspergillomarasmine A (AMA) is a natural Zn 2+ metallophore and inhibitor of metalloβ-lactamases (MBLs) which reverses β-lactam resistance. The first crystal structure of an AMA coordination complex is reported and reveals a pentadentate ligand with distorted octahedral geometry. We report the solid-phase synthesis of 23 novel analogs of AMA involving structural diversification of each subunit (L-Asp, L-APA1, and L-APA2). Inhibitory activity was evaluated in vitro using five strains of Escherichia coli producing globally prevalent MBLs. Further in vitro assessment was performed with purified recombinant enzymes and intracellular accumulation studies. Highly constrained structure−activity relationships were demonstrated, but three analogs revealed favorable characteristics where either Zn 2+ affinity or the binding mode to MBLs were improved. This study identifies compounds that can further be developed to produce more potent and broader-spectrum MBL inhibitors with improved pharmacodynamic/pharmacokinetic properties.

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