LRET-Based HTS of a Small-Compound Library for Inhibitors of Bacterial RNA Polymerase

Abstract

Resistance mechanisms against whole classes of antibiotics are currently developing faster than research generates new structurally different biologically active agents. The demand for new antimicrobial drugs has not been matched by the speed of discovery. The interface between sigma and core of bacterial RNA polymerase offers an attractive target for drug discovery, and we have previously described the development of a very robust high-throughput assay for this target based on luminescence resonance energy transfer. Here we describe a semiautomated screen of a commercially available library (Chembridge, San Diego, CA) that led to the identification of four small molecules, two of which have activity in preventing in vitro transcription and growth of Escherichia coli.

Identification of inhibitors of bacterial RNA polymerase

“…When testing compounds for their ability to inhibit the r-b 0 CH interaction, ELISAs were performed as above except the GST tagged wild-type CH-domain fragment (200 nM) was pre-mixed with 15 lM of the test compound at 37°C for 15 min before being added into the r A coated wells [21][22][23]. Compounds showing good levels of inhibition were further tested at a range of concentrations to determine preliminary K i values (Fig.…”

“…Therefore, despite many regions of r interacting with RNAP, only the contact between the CH-domain and r 2.2 needs to be prevented to abolish the formation of HE. Indeed, a high throughput screen has been developed that was designed to identify inhibitors of HE formation, and although several compounds were identified, the subsequent development of leads has not been reported [14,15].…”

Surface Plasmon Resonance – More Than a Screening Technology: Insights in the Binding Mode of σ 70 :Core Rnap Inhibitors

“…This binding site is highly conserved [18] and mutations occur only seldom since many transcription/σ-factors bind to this site on the β' subunit. Until today, very few small organic compounds have been described as σ 70 :core inhibitors such as SB2 [19], A5 [20] and GKL003 [21]. The inhibitors have been identified by experimental screening (ELISAbased assembly assay [19], LRET assay [20]), and computational methods (pharmacophore model [21]).…”

“…Until today, very few small organic compounds have been described as σ 70 :core inhibitors such as SB2 [19], A5 [20] and GKL003 [21]. The inhibitors have been identified by experimental screening (ELISAbased assembly assay [19], LRET assay [20]), and computational methods (pharmacophore model [21]). Although these compounds already show in vitro and in vivo activity, they have poor solubility and selectivity [20,22].…”

“…The inhibitors have been identified by experimental screening (ELISAbased assembly assay [19], LRET assay [20]), and computational methods (pharmacophore model [21]). Although these compounds already show in vitro and in vivo activity, they have poor solubility and selectivity [20,22]. In addition, the in vivo activity of GKL003 is very low with bacterial growth inhibition at only >1 mM equivalent to 660 μg/ml [21].…”