An investigation of New Delhi metallo-[beta]-lactamase : clinical variants, lipid modification & inhibition

Date

2017-05-12

Authors

Stewart, Alesha

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Abstract

Many β-lactam drugs, including penicillins, monobactams cephalosporins, and carbapenems, are used in clinical treatment of bacterial infections. However, resistant bacterial strains have surfaced, in part due to expression of β-lactam hydrolyzing enzymes called β-lactamases. One class of β-lactamases, metallo-β-lactamases (MBL), confers resistance to almost all clinically used bicyclic β-lactams through hydrolysis facilitated by active-site metal ions. The non-covalent mechanism of these enzymes enables them to escape the action of clinically used β-lactamase inhibitors, and there are currently no approved drugs that counteract their activity. One MBL that has caused particular alarm is New Delhi metallo-β-lactamase (NDM-1), that can be contracted through both hospital and communal settings. Clinically isolated bacteria strains that host NDM-1 have been reported to carry mutations in their bla[subscript NDM-1] gene sequence. The NDM-4, NDM-9, NDM-10*, and NDM-12 variants were characterized for zinc-binding and catalytic activity against a panel of structurally-diverse substrates. Unlike NDM-1, NDM-4 and NDM-12 (which share a M154L mutation) maintained high hydrolytic activity at low zinc concentrations for penam and carbapenem substrates. Furthermore, NDM-4 and NDM-12 were shown to have increased zinc affinity that may help maintain activity at low zinc concentrations similar to those found at infection sites. Full length NDM-1 has an N-terminal sequence that targets the transcribed enzyme to be transported to the periplasm where it binds Zn(II). The mature protein is lipid modified at the cysteine 26 position and embedded into the inner leaflet of the outer membrane. Transwell culture plates were used to examine if lipidation sequesters the enzyme and only provides resistance to the host bacterium (a "private good") in contrast to a β-lactamase lacking lipidation. Despite similar expression levels for enzyme variants, full-length NDM-1 does not protect neighboring cells against β-lactam treatment. However, the N-terminal deletion construct, which lacks the lipidation site, can effectively provide resistance to bystander cells (a "public good"). Finally, high-throughput screening assays were developed to analyze compounds and provide feedback that aided in the development and derivation of prospective NDM-1 inhibitors. There are no clinically-effective NDM-1 targeting drugs in use, so understanding the specificity and ligand-binding properties of this enzyme and its variants is important for the design of new drugs to counteract this global threat

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