1,4,7-Triazacyclononane Restores the Activity of β-Lactam Antibiotics against Metallo-β-Lactamase-ProducingEnterobacteriaceae: Exploration of Potential Metallo-β-Lactamase Inhibitors

Abstract

Metallo-β-lactamase (MBL)-producing Enterobacteriaceae are of grave clinical concern, particularly as there are no metallo-β-lactamase inhibitors approved for clinical use. The discovery and development of MBL inhibitors to restore the efficacy of available β-lactams are thus imperative. We investigated a zinc-chelating moiety, 1,4,7-triazacyclononane (TACN), for its inhibitory activity against clinical carbapenem-resistant Enterobacteriaceae. MICs, minimum bactericidal concentrations (MBCs), the serum effect, fractional inhibitory concentration indexes, and time-kill kinetics were determined using broth microdilution techniques according to Clinical and Laboratory Standards Institute (CSLI) guidelines. Enzyme kinetic parameters and the cytotoxic effects of TACN were determined using spectrophotometric assays. The interactions of the enzyme-TACN complex were investigated by computational studies. Meropenem regained its activity against carbapenemase-producing Enterobacteriaceae, with the MIC decreasing from between 8 and 64 mg/liter to 0.03 mg/liter in the presence of TACN. The TACN-meropenem combination showed bactericidal effects with an MBC/MIC ratio of ≤4, and synergistic activity was observed. Human serum effects on the MICs were insignificant, and TACN was found to be noncytotoxic at concentrations above the MIC values. Computational studies predicted that TACN inhibits MBLs by targeting their catalytic active-site pockets. This was supported by its inhibition constant (Ki), which was 0.044 μM, and its inactivation constant (Kinact), which was 0.0406 min−1, demonstrating that TACN inhibits MBLs efficiently and holds promise as a potential inhibitor. IMPORTANCE Carbapenem-resistant Enterobacteriaceae (CRE)-mediated infections remain a significant public health concern and have been reported to be critical in the World Health Organization’s priority pathogens list for the research and development of new antibiotics. CRE produce enzymes, such as metallo-β-lactamases (MBLs), which inactivate β-lactam antibiotics. Combination therapies involving a β-lactam antibiotic and a β-lactamase inhibitor remain a major treatment option for infections caused by β-lactamase-producing organisms. Currently, no MBL inhibitor–β-lactam combination therapy is clinically available for MBL-positive bacterial infections. Hence, developing efficient molecules capable of inhibiting these enzymes could be a promising way to overcome this phenomenon. TACN played a significant role in the inhibitory activity of the tested molecules against CREs by potentiating the activity of carbapenem. This study demonstrates that TACN inhibits MBLs efficiently and holds promises as a potential MBL inhibitor to help curb the global health threat posed by MBL-producing CREs.

Metallo-β-Lactamase Inhibitors Inspired on Snapshots from the Catalytic Mechanism

“…As NOTA was the most active compound, different analogues were synthetized and some of them ( 30 is an example) were able to restore the activity of meropenem toward carbapenem-resistant pathogens without showing toxicity against eukaryotic cells [ 247 ]. A related compound, 1,4,7-triazacyclononane (TACN), in combination with meropenem, showed bactericidal effects on different clinical Enterobacterales expressing B1 MBLs but it was shown to compromise the eukaryotic cell viability [ 248 ].…”

Epigenomics, genomics, resistome, mobilome, virulome and evolutionary phylogenomics of carbapenem-resistant Klebsiella pneumoniae clinical strains

“…Carbapenemases are capable of slightly and/or completely hydrolysing β-lactams, including 'last resort' carbapenems [27]. Class B carbapenemases, particularly bla NDM genes, have been reported to be more potent than the other groups and cannot be inhibited by commercially available β-lactamase inhibitors such as clavulanic acid, tazobactam or sulbactam [28,29]. However, metal chelators such as EDTA and mercaptopropionic acid are able to inhibit activity of class B carbapenemases [28].…”

The molecular effect of 1,4,7‐triazacyclononane on oxidative stress parameters in human hepatocellular carcinoma (HepG2) cells

“…[ 6 ] Resistance to these antibiotics has been achieved via a variety of ways including the production of β‐lactamases, which use hydrolytic pathways to break open the β‐lactam cyclic amide ring [ 5 ] as depicted in Figure 1, reduce membrane permeability and render the antibiotic ineffective. [ 7 ] Metallo‐β‐lactamases (MBLs) are class B enzymes that have been shown to be resistant to the last resort antibiotics such as meropenem and imipenem. [ 8–11 ] The MBLs are divided into three subclasses: B1, B2 and B3; which are defined by the difference in zinc coordination shell and how their amino acids are sequenced.…”

“…The inhibitor ( Figure 2), 1,4,7-tiazacyclononane (TACN), is a cyclic organic tridentate ligand with strong chelating abilities. [7] Metal complexes of TACN and its derivatives have been shown to form complexes that catalyse oxidative transformation, inhibit MBL-producing Enterobacteriaceae and restore the activity of β-lactam antibiotics with tolerable toxicity in HepG2 cells. [7,13] However, additional evidence is required for its clinical application.…”

“…[7] Metal complexes of TACN and its derivatives have been shown to form complexes that catalyse oxidative transformation, inhibit MBL-producing Enterobacteriaceae and restore the activity of β-lactam antibiotics with tolerable toxicity in HepG2 cells. [7,13] However, additional evidence is required for its clinical application. A series of TACN derivatives were synthesised and evaluated as iron (Fe 3+ ) depleting agents with the potential for use as anticancer therapy against human hepatocellular carcinoma.…”