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.

The Rac1 exchange factor Dock5 is essential for bone resorption by osteoclasts

“…(20,21) Briefly, yeasts were grown in 96-well plates in histidine-free and histidine-complemented dropout medium supplemented with 1% DMSO and 100 mM of one of the 2640 chemical compounds to be tested (ChemBridge, San Diego, CA, USA). (25) Growth curves were established by measuring OD600. Inhibitors were selected for their ability to inhibit selectively growth acceleration in histidine-free medium conferred by Dock5-DHR2 expression without affecting growth in histidinesupplemented medium.…”

Bacterial Transcription as a Target for Antibacterial Drug Development

“…The SB series (Fig. 2) was discovered by compound library screening against E. coli RNAP holoenzyme formation using an enzyme-linked immunosorbent assay (ELISA)-based assay (185). These compounds could inhibit E. coli 70 binding to RNAP with an IC 50 of 2 to 15 M, although their binding site was unknown.…”

Protein-protein Interactions: Network Analysis and Applications in Drug Discovery

“…An important bottleneck here is the poorly elucidated intra-bacterial as well as host-bacterial PPI networks, as will also be discussed later. It is not surprising that none of the antibiotics in clinical trials in 2011 are targeting PPIs [49], as only one reported HTS aimed at identifying bacterial PPI disruptors [50]. The essential role of RNA polymerase for bacterial growth, the conservation of the interface between σ factors and the core polymerase in bacteria, and the absence of σ factors in mammalian cells motivated André and coworkers to identify specific inhibitors of core-σ interaction.…”

“…The essential role of RNA polymerase for bacterial growth, the conservation of the interface between σ factors and the core polymerase in bacteria, and the absence of σ factors in mammalian cells motivated André and coworkers to identify specific inhibitors of core-σ interaction. Using an in vitro HT ELISA, they identified synthetic small molecule compounds (SB2 and SB8) that inhibit this interaction with an IC 50 in the low micromolar range. These molecules were shown to be bactericidal for a wide range of Gram-positive bacteria such as (multidrug-resistant) S. aureus, S. epidermidis, B. cereus, B. anthracis, S. pneumoniae as well as some Gram-negative bacteria such as Bacteroides spp, but were unfortunately inactive against E. coli or P. aeruginosa due to low bacterial entry of the compounds [50,51].…”