Binding Mode Characterization of Novel RNA Polymerase Inhibitors Using a Combined Biochemical and NMR Approach

Bacterial
RNA polymerase (RNAP) represents a validated target for the development
of broad-spectrum antibiotics. However, the medical value of RNAP
inhibitors in clinical use is limited by the prevalence of resistant
strains. To overcome this problem, we focused on the exploration of
alternative target sites within
the RNAP. Previously, we described the discovery of a novel RNAP inhibitor
class containing an ureidothiophene-2-carboxylic acid core structure.
Herein, we demonstrate that these compounds are potent against a set
of methicillin-resistant Staphylococcus aureus (MRSA)
strains (MIC 2–16 μg mL–1) and rifampicin-resistant Escherichia coli TolC strains (MIC 12.5–50 μg
mL–1). Additionally, an abortive transcription assay
revealed that these compounds inhibit the bacterial transcription
process during the initiation phase. Furthermore, the binding mode
of the ureidothiophene-2-carboxylic acids was characterized by mutagenesis
studies and ligand-based NMR spectroscopy. Competition saturation
transfer difference (STD) NMR experiments with the described RNAP
inhibitor myxopyronin A (Myx) suggest that the ureidothiophene-2-carboxylic
acids compete with Myx for the same binding site in the
RNAP switch region. INPHARMA (interligand NOE for pharmacophore mapping)
experiments and molecular docking simulations provided a binding model
in which the ureidothiophene-2-carboxylic acids occupy the region
of the Myx western chain binding site and slightly occlude
that of the eastern chain. These results demonstrate that the ureidothiophene-2-carboxylic
acids are a highly attractive new class of RNAP inhibitors that can
avoid the problem of resistance.