title: A multiwell assay to isolate compounds inhibiting the assembly of the prokaryotic RNA polymerase.
abstract: We have developed a multiwell assay for the detection of modulators of prokaryotic transcription based on the quantification of protein-protein interaction. This assay consists of three steps: (a) the immobilization of the Escherichia coli protein sigma70 in the well, (b) the incubation of the immobilized protein with core RNA polymerase and a potential inhibitor, and (c) washing and quantification of the binding of core to sigma70 with a monoclonal antibody conjugated to horseradish peroxidase. We show that this assay is sensitive, reproducible, and robust, and is able to discriminate between control competitors with different affinities. We demonstrate the usefulness of the assay to screen for microbial RNA polymerase inhibitors as potential new drugs for the treatment of emerging antibiotic-resistant bacteria.
authors.affiliation: CNRS UMR 5160 Faculté de Pharmacie, Montpellier, France.
authors.forename: Estelle
authors.initials: E
authors.lastname: André
authors.forename: Lionel
authors.initials: L
authors.lastname: Bastide
authors.forename: Philippe
authors.initials: P
authors.lastname: Villain-Guillot
authors.forename: Jaqueline
authors.initials: J
authors.lastname: Latouche
authors.forename: Joelle
authors.initials: J
authors.lastname: Rouby
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Leonetti
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000494:pharmacology; D002850:Chromatography, Gel; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000187:drug effects* / Q000378:metabolism*; D015723:Gene Library; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000494:pharmacology; D015203:Reproducibility of Results; D012808:Sigma Factor / Q000378:metabolism; D012997:Solvents; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D012808:Sigma Factor; D012997:Solvents; D006735:Horseradish Peroxidase; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases