A multitarget assay for inhibitors of membrane-associated steps of peptidoglycan biosynthesis.
Peptidoglycan synthesis begins in the cytoplasm with the condensation of UDP-N-acetyl glucosamine (UDP-GlcNAc) and phosphoenolpyruvate catalyzed by UDP-N-acetylglucosamine enolpyruvoyl transferase. UDP-GlcNAc is also utilized as substrate for the glycosyltransferase MurG, a membrane-bound enzyme that catalyzes the production of lipid II. Membranes from Escherichia coli cells overproducing MurG support peptidoglycan formation at a rate approximately fivefold faster than membranes containing wild-type levels of MurG. Conditions have been optimized for the production of large amounts of membranes with increased levels of MurG, allowing the development of an assay suitable for high-throughput screening of large compound libraries. The quality of the purified membranes was assessed by electron microscopy and also by testing cross-linked peptidoglycan production. Moreover, kinetic studies allowed the determination of optimal concentrations of the substrates and membranes to be utilized for maximum sensitivity of the assay. Using a 96-well assay format, the IC50 values for vancomycin, tunicamycin, flavomycin, and bacitracin were 1.1 microM, 0.01 microg/ml, 0.03 microg/ml, and 0.7 microg/ml, respectively.
Barbosa
Maria D F S
MD
Department of Antimicrobial Research, Bristol-Myers Squibb Pharmaceuticals, Wilmington, Delaware 19880, USA. barbosamdf@aol.com
Ross
Harold O
HO
Hillman
Milton C
MC
Meade
Raymond P
RP
Kurilla
Michael G
MG
Pompliano
David L
DL
D000900:Anti-Bacterial Agents / Q000032:analysis / Q000494:pharmacology*; D001425:Bacterial Outer Membrane Proteins; D001681:Biological Assay / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D020128:Inhibitory Concentration 50; D007700:Kinetics; D017351:N-Acetylglucosaminyltransferases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D010457:Peptidoglycan / Q000096:biosynthesis*; D015203:Reproducibility of Results; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D010457:Peptidoglycan; D017351:N-Acetylglucosaminyltransferases; C064057:UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide)pyrophosphoryl-undecaprenol N-acetylglucosamine transferase