title: Targeting the MraY and MurG bacterial enzymes for antimicrobial therapeutic intervention.
abstract: 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.
authors.affiliation: Pharmaceutical Research Institute, Bristol-Myers Squibb, Wallingford, CT 06492-7660, USA.
authors.forename: Laura E
authors.initials: LE
authors.lastname: Zawadzke
authors.forename: Ping
authors.initials: P
authors.lastname: Wu
authors.forename: Lynda
authors.initials: L
authors.lastname: Cook
authors.forename: Li
authors.initials: L
authors.lastname: Fan
authors.forename: Margaret
authors.initials: M
authors.lastname: Casperson
authors.forename: Mona
authors.initials: M
authors.lastname: Kishnani
authors.forename: Deepa
authors.initials: D
authors.lastname: Calambur
authors.forename: Sandra J
authors.initials: SJ
authors.lastname: Hofstead
authors.forename: Ramesh
authors.initials: R
authors.lastname: Padmanabha
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020128:Inhibitory Concentration 50; D015394:Molecular Structure; D008970:Molecular Weight; D017351:N-Acetylglucosaminyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009705:Nucleosides / Q000494:pharmacology; D013997:Time Factors; D014166:Transferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017855:Transferases (Other Substituted Phosphate Groups)
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D009705:Nucleosides; C059803:mureidomycin A; D014166:Transferases; D017351:N-Acetylglucosaminyltransferases; C064057:UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide)pyrophosphoryl-undecaprenol N-acetylglucosamine transferase; D017855:Transferases (Other Substituted Phosphate Groups); C079803:mraY protein, Bacteria