3.2.2. Inhibitors of the PhoP-DNA Complex

Three active compounds (NCGC00093547, NCGC00244580 and NCGC00161636) that directly bind to PhoP and therefore inhibit PhoP-DNA interactions were found in a screening assay based on Foster resonance energy transfer (FRET). For this screening a DNA-Protein complex of Cy3-labeled DNA and Cy5-labeled PhoP protein was exposed to compounds of interest. Inhibitors of this DNA-Protein complex led to dissociation and consequently to a reduced FRET signal [110]. Direct binding of inhibitors to PhoP was confirmed by thermal shift assays in which target-bound inhibitors stabilize the protein and increase the melting temperature, which can be quantified using the fluorescence signal of fluorophore-protein complexes. Compounds NCGC00093547 and NCGC00161636 increased PhoP melting temperature by 14 ◦C and 18 ◦C with an IC50 of 15.6 and 15.5 μM, respectively. Data on in vivo or ex vivo activity of these compounds is not available yet.
