A multiwell assay to isolate compounds inhibiting the assembly of the prokaryotic RNA polymerase.
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.
André
Estelle
E
CNRS UMR 5160 Faculté de Pharmacie, Montpellier, France.
Bastide
Lionel
L
Villain-Guillot
Philippe
P
Latouche
Jaqueline
J
Rouby
Joelle
J
Leonetti
Jean-Paul
JP
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
D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D012808:Sigma Factor; D012997:Solvents; D006735:Horseradish Peroxidase; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases