Methods to identify and characterize inhibitors of bacterial RNA polymerase.
RNA polymerase is essential to the viability of bacteria in all phases of growth and development and is a proven chemotherapeutic target as the cellular target of the rifamycin class of antibiotics. However, despite the characterization of multiple different classes of natural products that selectively target bacterial RNA polymerase, and the identification of a limited number of synthetic compound inhibitors, only agents of the rifamycin class have been developed and approved for human clinical use as antibiotics. Herein we describe a scintillation proximity assay (SPA) for identifying and characterizing inhibitors of bacterial RNA polymerases and that is applicable to de novo drug discovery programs through application of automated high-throughput screening methods. In addition, we describe gel electrophoresis-based methods that are applicable to the detailed characterization of inhibitors of transcriptional initiation or elongation by bacterial RNA polymerases.
Lynch
A Simon
AS
Cumbre Pharmaceuticals Inc., Dallas, TX, USA.
Du
Qun
Q
D001331:Automation; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D004353:Drug Evaluation, Preclinical; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D012588:Scintillation Counting / Q000379:methods*; D012808:Sigma Factor / Q000302:isolation & purification; D013211:Staphylococcus aureus / Q000201:enzymology; D014316:Tritium / Q000032:analysis
D004791:Enzyme Inhibitors; D012808:Sigma Factor; D014316:Tritium; D012321:DNA-Directed RNA Polymerases