Establishment of an In Bacterio Assay for the Assessment of Carbon Storage Regulator A (CsrA) Inhibitors.

Polymicrobial infections involving various combinations of microorganisms, such as Escherichia, Pseudomonas, or Yersinia, can lead to acute and chronic diseases in for example the gastrointestinal and respiratory tracts. Our aim is to modulate microbial communities by targeting the posttranscriptional regulator system called carbon storage regulator A (CsrA) (or also repressor of secondary metabolites (RsmA)). In previous studies, we identified easily accessible CsrA binding scaffolds and macrocyclic CsrA binding peptides through biophysical screening and phage display technology. However, due to the lack of an appropriate in bacterio assay to evaluate the cellular effects of these inhibitor hits, the focus of the present study is to establish an in bacterio assay capable of probing and quantifying the impact on CsrA-regulated cellular mechanisms. We have successfully developed an assay based on a luciferase reporter gene assay, which in combination with a qPCR expression gene assay, allows for the monitoring of expression levels of different downstream targets of CsrA. The chaperone protein CesT was used as a suitable positive control for the assay, and in time-dependent experiments, we observed a CesT-mediated increase in bioluminescence over time. By this means, the cellular on-target effects of non-bactericidal/non-bacteriostatic virulence modulating compounds targeting CsrA/RsmA can be evaluated.

Phage display-based discovery of cyclic peptides against the broad spectrum bacterial anti-virulence target CsrA.

Small macrocyclic peptides are promising candidates for new anti-infective drugs. To date, such peptides have been poorly studied in the context of anti-virulence targets. Using phage display and a self-designed peptide library, we identified a cyclic heptapeptide that can bind the carbon storage regulator A (CsrA) from Yersinia pseudotuberculosis and displace bound RNA. This disulfide-bridged peptide, showed an IC50 value in the low micromolar range. Upon further characterization, cyclisation was found to be essential for its activity. To increase metabolic stability, a series of disulfide mimetics were designed and a redox-stable 1,4-disubstituted 1,2,3-triazole analogue displayed activity in the double-digit micromolar range. Further experiments revealed that this triazole peptidomimetic is also active against CsrA from Escherichia coli and RsmA from Pseudomonas aeruginosa. This study provides an ideal starting point for medicinal chemistry optimization of this macrocyclic peptide and might pave the way towards broad-acting virulence modulators.

First Small-Molecule Inhibitors Targeting the RNA-Binding Protein IGF2BP2/IMP2 for Cancer Therapy.

The RNA-binding protein IGF2BP2/IMP2/VICKZ2/p62 is overexpressed in several tumor entities, promotes tumorigenesis and tumor progression, and has been suggested to worsen the disease outcome. The aim of this study is to (I) validate IMP2 as a potential target for colorectal cancer, (II) set up a screening assay for small-molecule inhibitors of IMP2, and (III) test the biological activity of the obtained hit compounds. Analyses of colorectal and liver cancer gene expression data showed reduced survival in patients with a high IMP2 expression and in patients with a higher IMP2 expression in advanced tumors. In vitro target validation in 2D and 3D cell cultures demonstrated a reduction in cell viability, migration, and proliferation in IMP2 knockout cells. Also, xenotransplant tumor cell growth in vivo was significantly reduced in IMP2 knockouts. Different compound libraries were screened for IMP2 inhibitors using a fluorescence polarization assay, and the results were confirmed by the thermal shift assay and saturation-transfer difference NMR. Ten compounds, which belong to two classes, that is, benzamidobenzoic acid class and ureidothiophene class, were validated in vitro and showed a biological target specificity. The three most active compounds were also tested in vivo and exhibited reduced tumor xenograft growth in zebrafish embryos. In conclusion, our findings support that IMP2 represents a druggable target to reduce tumor cell proliferation.