This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Barbosa, M. D. F. S. Articles by Pompliano, D. L. Search for Related Content PubMed PubMed Citation Articles by Barbosa, M. D. F. S. Articles by Pompliano, D. L.

Next Article 

Antimicrobial Agents and Chemotherapy, April 2002, p. 943-946, Vol. 46, No. 4
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.4.943-946.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Development of a Whole-Cell Assay for Peptidoglycan Biosynthesis Inhibitors

Department of Antimicrobial Research, Bristol-Myers Squibb Pharma, Wilmington, Delaware 19880

Received 10 September 2001/ Returned for modification 31 October 2001/ Accepted 31 December 2001

Osmotically stabilized Escherichia coli cells subjected to freezing and thawing were utilized as the source of enzymes for a peptidoglycan pathway assay that can be used to simultaneously test all targets of the committed steps of cell wall biosynthesis. The use of ¹⁴C-labeled UDP-N-acetylglucosamine (UDP-GlcNAc) as a substrate allows the direct detection of cross-linked peptidoglycan formed. The assay was validated with known antibiotics. Fosfomycin was the strongest inhibitor of the pathway assay, with a 50% inhibitory concentration of 1 µM. Flavomycin, bacitracin, vancomycin, D-cycloserine, penicillin G, and ampicillin also inhibited formation of radiolabeled peptidoglycan by the E. coli cells. Screening of compounds identified two inhibitors of the pathway, Cpd1 and Cpd2. Subsequent tests with a biochemical assay utilizing purified enzyme implicated UDP-GlcNAc enolpyruvyl transferase (MurA) as the target of Cpd1. This compound inhibits the first enzyme of the pathway in a time-dependent manner. Moreover, enzyme inactivation is dependent on preincubation in the presence of UDP-GlcNAc, which forms a complex with MurA, exposing its active site. Cpd1 also displayed antimicrobial activity against a panel of microorganisms. The pathway assay used in conjunction with assays for individual enzymes provides an efficient means of detecting and characterizing novel antimicrobial agents.

This article has been cited by other articles:

• Baum, E. Z., Crespo-Carbone, S. M., Abbanat, D., Foleno, B., Maden, A., Goldschmidt, R., Bush, K. (2006). Utility of Muropeptide Ligase for Identification of Inhibitors of the Cell Wall Biosynthesis Enzyme MurF. Antimicrob. Agents Chemother. 50: 230-236 [Abstract] [Full Text]  
• Price, N. P., Momany, F. A. (2005). Modeling bacterial UDP-HexNAc: polyprenol-P HexNAc-1-P transferases. Glycobiology 15: 29R-42R [Abstract] [Full Text]  
• Chandrakala, B., Shandil, R. K., Mehra, U., Ravishankar, S., Kaur, P., Usha, V., Joe, B., deSousa, S. M. (2004). High-Throughput Screen for Inhibitors of Transglycosylase and/or Transpeptidase Activities of Escherichia coli Penicillin Binding Protein 1b. Antimicrob. Agents Chemother. 48: 30-40 [Abstract] [Full Text]