Discovery of the first small-molecule CsrA-RNA interaction inhibitors using biophysical screening technologies.

AIM: CsrA is a global post-transcriptional regulator protein affecting mRNA translation and/or stability. Widespread among bacteria, it is essential for their full virulence and thus represents a promising anti-infective drug target. Therefore, we aimed at the discovery of CsrA-RNA interaction inhibitors. Results & methodology: We followed two strategies: a screening of small molecules (A) and an RNA ligand-based approach (B). Using surface plasmon resonance-based binding and fluorescence polarization-based competition assays, (A) yielded seven small-molecule inhibitors, among them MM14 (IC50 of 4 µM). (B) resulted in RNA-based inhibitor GGARNA (IC50 of 113 µM). CONCLUSION: The first small-molecule inhibitors of the CsrA-RNA interaction were discovered exhibiting micromolar affinities. These hits represent tools to investigate the effects of CsrA-RNA interaction inhibition on bacterial virulence.

Treatment effect on biases in size estimation in spider phobia.

BACKGROUND: The current study investigates biases in size estimations made by spider-phobic and healthy participants before and after treatment. METHOD: Forty-one spider-phobic and 20 healthy participants received virtual reality (VR) exposure treatment and were then asked to rate the size of a real spider immediately before and, on average, 15days after the treatment. During the VR exposure treatment skin conductance response was assessed. RESULTS: Prior to the treatment, both groups tended to overestimate the size of the spider, but this size estimation bias was significantly larger in the phobic group than in the control group. The VR exposure treatment reduced this bias, which was reflected in a significantly smaller size rating post treatment. However, the size estimation bias was unrelated to the skin conductance response. CONCLUSION: Our results confirm the hypothesis that size estimation by spider-phobic patients is biased. This bias is not stable over time and can be decreased with adequate treatment.

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.

Peptide-based investigation of the Escherichia coli RNA polymerase σ(70):core interface as target site.

The number of bacterial strains that are resistant against antibiotics increased dramatically during the past decades. This fact stresses the urgent need for the development of new antibacterial agents with novel modes of action targeting essential enzymes such as RNA polymerase (RNAP). Bacterial RNAP is a large multi-subunit complex consisting of a core enzyme (subunits: α(2)ßß'ω) and a dissociable sigma factor (σ(70); holo enzyme: α(2)ßß'ωσ(70)) that is responsible for promoter recognition and transcription initiation. The interface between core RNAP and σ(70) represents a promising binding site. Nevertheless, detailed studies investigating its druggability are rare. Compounds binding to this region could inhibit this protein-protein interaction and thus holo enzyme formation, resulting in inhibition of transcription initiation. Sixteen peptides covering different regions of the Escherichia coli σ(70):core interface were designed; some of them-all derived from σ(70) 2.2 region-led to a strong RNAP inhibition. Indeed, an ELISA-based experiment confirmed the most active peptide P07 to inhibit the σ(70):core interaction. Furthermore, an abortive transcription assay revealed that P07 impedes transcription initiation. In order to study the mechanism of action of P07 in more detail, molecular dynamics simulations and a rational amino acid replacement study were performed, leading to the conclusion that P07 binds to the coiled-coil region in ß' and that its flexible N-terminus inhibits the enzyme by interaction with the ß' lid-rudder-system (LRS). This work revisits the ß' coiled-coil as a hot spot for the protein-protein interaction inhibition and expands it by introduction of the LRS as target site.