Efficacy of aspergillomarasmine A/meropenem combinations with and without avibactam against bacterial strains producing multiple ß-lactamases.

The effectiveness of ß-lactam antibiotics is increasingly threatened by resistant bacteria that harbor hydrolytic ß-lactamase enzymes. Depending on the class of ß-lactamase present, ß-lactam hydrolysis can occur through one of two general molecular mechanisms. Metallo-ß-lactamases (MBLs) require active site Zn2+ ions, whereas serine-ß-lactamases (SBLs) deploy a catalytic serine residue. The result in both cases is drug inactivation via the opening of the ß-lactam warhead of the antibiotic. MBLs confer resistance to most ß-lactams and are non-susceptible to SBL inhibitors, including recently approved diazabicyclooctanes, such as avibactam; consequently, these enzymes represent a growing threat to public health. Aspergillomarasmine A (AMA), a fungal natural product, can rescue the activity of the ß-lactam antibiotic meropenem against MBL-expressing bacterial strains. However, the effectiveness of this ß-lactam/ß-lactamase inhibitor combination against bacteria producing multiple ß-lactamases remains unknown. We systematically investigated the efficacy of AMA/meropenem combination therapy with and without avibactam against 10 Escherichia coli and 10 Klebsiella pneumoniae laboratory strains tandemly expressing single MBL and SBL enzymes. Cell-based assays demonstrated that laboratory strains producing NDM-1 and KPC-2 carbapenemases were resistant to the AMA/meropenem combination but became drug-susceptible upon adding avibactam. We also probed these combinations against 30 clinical isolates expressing multiple ß-lactamases. E. coli, Enterobacter cloacae, and K. pneumoniae clinical isolates were more susceptible to AMA, avibactam, and meropenem than Pseudomonas aeruginosa and Acinetobacter baumannii isolates. Overall, the results demonstrate that a triple combination of AMA/avibactam/meropenem has potential for empirical treatment of infections caused by multiple ß-lactamase-producing bacteria, especially Enterobacterales.

Aspergillomarasmine A inhibits metallo-ß-lactamases by selectively sequestering Zn2.

Class B metallo-ß-lactamases (MBLs) are Zn2+-dependent enzymes that catalyze the hydrolysis of ß-lactam antibiotics to confer resistance in bacteria. Several problematic groups of MBLs belong to subclass B1, including the binuclear New Delhi MBL (NDM), Verona integrin-encoded MBL, and imipenemase-type enzymes, which are responsible for widespread antibiotic resistance. Aspergillomarasmine A (AMA) is a natural aminopolycarboxylic acid that functions as an effective inhibitor of class B1 MBLs. The precise mechanism of action of AMA is not thoroughly understood, but it is known to inactivate MBLs by removing one catalytic Zn2+ cofactor. We investigated the kinetics of MBL inactivation in detail and report that AMA is a selective Zn2+ scavenger that indirectly inactivates NDM-1 by encouraging the dissociation of a metal cofactor. To further investigate the mechanism in living bacteria, we used an active site probe and showed that AMA causes the loss of a Zn2+ ion from a low-affinity binding site of NDM-1. Zn2+-depleted NDM-1 is rapidly degraded, contributing to the efficacy of AMA as a ß-lactam potentiator. However, MBLs with higher metal affinity and stability such as NDM-6 and imipenemase-7 exhibit greater tolerance to AMA. These results indicate that the mechanism of AMA is broadly applicable to diverse Zn2+ chelators and highlight that leveraging Zn2+ availability can influence the survival of MBL-producing bacteria when they are exposed to ß-lactam antibiotics.

Suppression of ß-Lactam Resistance by Aspergillomarasmine A Is Influenced by both the Metallo-ß-Lactamase Target and the Antibiotic Partner.

The rise of Gram-negative pathogens expressing metallo-ß-lactamases (MBLs) is a growing concern, threatening the efficacy of ß-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 ß-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/ß-lactam combinations did not correlate with outer membrane permeability or drug efflux. We determined that the optimal ß-lactam partners for AMA are the carbapenem antibiotics and that the efficacy of AMA is linked to the Zn2+ affinity of specific MBLs.

Arginine-containing peptides as potent inhibitors of VIM-2 metallo-ß-lactamase.

BACKGROUND: Metallo-ß-lactamases (MBLs) play an important role in the emergence of microbial resistance to ß-lactam antibiotics, and are hence considered targets for the design of novel therapeutics. We here report on the inhibitory effect of peptides containing multiple arginine residues on VIM-2, a clinically important MBL from Pseudomonas aeruginosa. METHODS: Enzyme kinetic assays in combination with fluorescence spectroscopy and stopped-flow UV-Vis spectrophotometry were utilized to explore the structure-activity relationship of peptides as inhibitors of VIM-2. RESULTS: Our studies show that the inhibitory potency of the investigated peptides was mainly dependent on the number of arginine residues in the center of the peptide sequence, and on the composition of the N-terminus. The most potent inhibitors were found to curtail enzyme function in the mid-to-low nanomolar range. Salts generally reduced peptide-mediated inhibition. Analysis of the mode of inhibition suggests the peptides to act as mixed-type inhibitors with a higher affinity for the enzyme-substrate complex. Stopped-flow UV-Vis and fluorescence studies revealed the peptides to induce rapid protein aggregation, a phenomenon strongly correlated to the peptides' inhibitory potency. Inhibition of IMP-1 (another subclass B1 MBL) by the peptides was found to be much weaker than that observed with VIM-2, a finding which might be related to subtle molecular differences in the protein surfaces. CONCLUSION: The reported data indicate that arginine-containing peptides can serve as potent, aggregation-inducing inhibitors of VIM-2, and potentially of other MBLs. GENERAL SIGNIFICANCE: Arginine-containing peptides can be considered as a novel type of potent MBL inhibitors.

Three-Dimensional Structure and Optimization of the Metallo-ß-Lactamase Inhibitor Aspergillomarasmine A.

The aminopolycarboxylic acid aspergillomarasmine A (AMA) is a natural Zn2+ metallophore and inhibitor of metallo-ß-lactamases (MBLs) which reverses ß-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 Zn2+ 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.

Inhibitors of metallo-ß-lactamases.

The ß-lactams are the most successful class of antibiotic drugs but they are vulnerable to inactivation by a growing cadre of ß-lactamases that now number more than a thousand variants. ß-Lactamases operate by one of two general chemical mechanisms either catalyzing ß-lactam ring hydrolysis via a covalent enzyme intermediate through the aegis of an active site serine residue or through a noncovalent Zn-dependent mechanism. The Ser-ß-lactamases are currently dominant in the clinic and consequently, there has been great effort to identify inhibitors and to co-formulate these with ß-lactam antibiotics. Four such inhibitors are approved for human clinical use and several more are in clinical trials. Metallo-ß-lactamases (MBLs), on the other hand, are only now emerging as a global threat and consequently, inhibitor discovery has lagged behind their Ser counterparts. There are now several examples of MBL inhibitors that operate either in a Zn-dependent or Zn-independent mode. The Zn-dependent compounds are more prevalent and some show efficacy in animal models of infection. These compounds function by either acting as an alternate metal ligand, usually displacing a jointly held hydroxide ion shared by enzymes with two Zn2+ ions, or alternately by striping Zn from the active site. The increase in the number of candidate MBL inhibitors over recent years reflects the growing clinical challenge of pathogens expressing these enzymes that result in a carbapenem resistance phenotype. While none of these inhibitors are yet in human clinical trials, the increasing importance of these enzymes in drug failure is a strong incentive to continue identifying and characterizing such molecules.