title: A high-throughput screen of the GTPase activity of Escherichia coli EngA to find an inhibitor of bacterial ribosome biogenesis.
abstract: The synthesis of ribosomes is an essential process, which is aided by a variety of trans-acting factors in bacteria. Among these is a group of GTPases essential for bacterial viability and emerging as promising targets for new antibacterial agents. Herein, we describe a robust high-throughput screening process for inhibitors of one such GTPase, the Escherichia coli EngA protein. The primary screen employed an assay of phosphate production in a 384-well density. Reaction conditions were chosen to maximize sensitivity for the discovery of competitive inhibitors while maintaining a strong signal amplitude and low noise. In a pilot screen of 31,800 chemical compounds, 44 active compounds were identified. Furthermore, we describe the elimination of nonspecific inhibitors that were detergent sensitive or reactive as well as those that interfered with the high-throughput phosphate assay. Four inhibitors survived these common counterscreens for nonspecificity, but these chemicals were also inhibitors of the unrelated enzyme dihydrofolate reductase, suggesting that they too were promiscuously active. The high-throughput screen of the EngA protein described here provides a meticulous pilot study in the search for specific inhibitors of GTPases involved in ribosome biogenesis.
authors.affiliation: 1M. G. DeGroote Institute for Infectious Disease Research and Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada.
authors.forename: Amrita
authors.initials: A
authors.lastname: Bharat
authors.forename: Jan E
authors.initials: JE
authors.lastname: Blanchard
authors.forename: Eric D
authors.initials: ED
authors.lastname: Brown
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry*; D004353:Drug Evaluation, Preclinical; D057075:Enzyme Assays; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D057166:High-Throughput Screening Assays; D015203:Reproducibility of Results; D054681:Ribosome Subunits, Large, Bacterial / Q000378:metabolism; D054680:Ribosome Subunits, Small, Bacterial / Q000378:metabolism; D059629:Signal-To-Noise Ratio
chemical_list: D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; C541027:EngA protein, E coli; D019204:GTP-Binding Proteins
keywords: EngA; GTPase; enzyme screen; ribosome biogenesis