Targeting the MraY and MurG bacterial enzymes for antimicrobial therapeutic intervention

Assays for two enzymes from Escherichia coli were developed and validated as antibacterial inhibitor screens. The MraY and MurG enzymes were overexpressed and purified as the membrane fraction or to homogeneity, respectively. The MurG enzyme was expressed with a six-histidine tag using an optimized minimal-medium protocol for subsequent purification. Although traditional assays were established, the enzymes were also assayed via a 96-well membrane plate assay and a 384-well scintillation proximity-based assay developed herein. These assays afford a more economical and high-throughput evaluation of inhibitors. A mureidomycin inhibitor mix was used as a control for the assay development and screen validation. Several inhibitors resulting from a high-throughput screen were found and evaluated for potential therapeutic use.