Antimicrobial triazinedione inhibitors of the translocase MraY–protein E interaction site: synergistic effects with bacitracin imply a new mechanism of action

Abstract

Escherichia coli translocase MraY is the target for bacteriolytic protein E from bacteriophage ϕX174, interacting at a site close to Phe-288 on helix 9, on the extracellular face of the protein. A peptide motif Arg-Trp-x-x-Trp from protein E was used to design a set of triazinedione peptidomimetics, which inhibit particulate MraY (6d IC₅₀ 48 μM), and show antimicrobial activity against Gram-negative and Gram-positive antibiotic-resistant clinical strains (7j MIC Acinetobacter baumannii 16 μg mL⁻¹, Staphyloccoccus aureus MRSA 2–4 μg mL⁻¹). Docking against a predicted structure for E. coli MraY revealed two possible binding sites close to helix 9, the binding site for protein E. Antimicrobial activity of analogue 6j was found to be synergistic with bacitracin in Micrococcus flavus, consistent with a link between this inhibition site and undecaprenyl phosphate uptake. Alkaloid michellamine B, also predicted to bind in the cleft adjacent to helix 9, was also found to be synergistic with bacitracin. These data provide experimental evidence that the unusual hydrophobic cleft adjacent to helix 9 in MraY is involved in uptake of undecaprenyl phosphate, in addition to recently identified transporters UptA and PopT, and that this process can be targeted by small molecules as a novel antibacterial mechanism.