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Antimicrobial Agents and Chemotherapy, June 2009, p. 2306-2311, Vol. 53, No. 6
0066-4804/09/$08.00+0     doi:10.1128/AAC.01572-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

High-Throughput Screening Identifies Novel Inhibitors of the Acetyltransferase Activity of Escherichia coli GlmU{triangledown}

Mark P. Pereira,1 Jan E. Blanchard,2 Cecilia Murphy,2 Steven L. Roderick,3 and Eric D. Brown1,2*

Department of Biochemistry and Biomedical Sciences and Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario L8N 3Z5, Canada,1 McMaster High Throughput Screening Laboratory, Department of Biochemistry, McMaster University, Hamilton, Ontario L8N 3Z5, Canada,2 Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 104613

Received 25 November 2008/ Returned for modification 2 January 2009/ Accepted 27 March 2009

The bifunctional GlmU protein catalyzes the formation of UDP-N-acetylglucosamine in a two-step reaction using the substrates glucosamine-1-phosphate, acetyl coenzyme A, and UTP. This metabolite is a common precursor to the synthesis of bacterial cell surface carbohydrate polymers, such as peptidoglycan, lipopolysaccharide, and wall teichoic acid that are involved in the maintenance of cell shape, permeability, and virulence. The C-terminal acetyltransferase domain of GlmU exhibits structural and mechanistic features unique to bacterial UDP-N-acetylglucosamine synthases, making it an excellent target for antibacterial design. In the work described here, we have developed an absorbance-based assay to screen diverse chemical libraries in high throughput for inhibitors to the acetyltransferase reaction of Escherichia coli GlmU. The primary screen of 50,000 drug-like small molecules identified 63 hits, 37 of which were specific to acetyltransferase activity of GlmU. Secondary screening and mode-of-inhibition studies identified potent inhibitors where compound binding within the acetyltransferase active site was requisite on the presence of glucosamine-1-phosphate and were competitive with the substrate acetyl coenzyme A. These molecules may represent novel chemical scaffolds for future antimicrobial drug discovery. In addition, this work outlines the utility of catalytic variants in targeting specific activities of bifunctional enzymes in high-throughput screens.

* Corresponding author. Mailing address: Department of Biochemistry and Biomedical Sciences and Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada. Phone: (905) 525-9140, ext. 22454. Fax: (905) 522-9033. E-mail: ebrown{at}mcmaster.ca

{triangledown} Published ahead of print on 6 April 2009.

Antimicrobial Agents and Chemotherapy, June 2009, p. 2306-2311, Vol. 53, No. 6
0066-4804/09/$08.00+0     doi:10.1128/AAC.01572-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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