Targeting the Moonlighting Chaperone Function of PQS Response Protein PqsE

The increasing spread of multidrug resistant pathogens such as Pseudomonas aeruginosa necessitates the development of novel therapeutic strategies. Since P. aeruginosa exhibits its pathogenicity largely through the production of virulence factors, virulence factor inhibitors, also referred to as pathoblockers, represent a promising approach. In contrast to conventional antibiotics, pathoblockers do not kill the bacteria, but attenuate their virulence. The quorum sensing system plays a central role in the regulation of these virulence factors. Of particular interest is the protein-protein interaction between the transcription factor RhlR and the thioesterase PqsE, as this interaction is unique to P. aeruginosa. It contributes significantly to the regulation of numerous virulence-associated genes and therefore represents an attractive target for the development of virulence factor inhibitors. In contrast to previously described virulence factor inhibitors that act only on individual virulence factors, the PqsE-RhlR interaction is considered a promising target. The aim of this study was to identify and characterize ligands that inhibit the PqsE-RhlR interaction, providing a foundation for the development of novel virulence factor inhibitors. To circumvent the instability of the RhlR wild type, a stabilized mutant, RhlR-P37 was engineered and characterized, demonstrating functional equivalence to the wild type in terms of its interaction with PqsE. A Förster resonance energy transfer-based assay was established to reliably detect the PqsE-RhlR-P37 interaction and employed in a single-dose screen of over 30000 ligands. Compounds that significantly repressed the assay signal were further evaluated through a cascade of analyses, including dose-response experiments, an orthogonal competitive microscale thermophoresis screen, and in cellulo tests for pyocyanin reduction. Due to limited sample availability, selected ligands were also tested in a miniaturized in cellulo pyocyanin assay. Of the 30000 compounds initially screened, 21 were found to inhibit pyocyanin production in P. aeruginosa. These active ligands were then subjected to additional biophysical and structural analyses to elucidate their interactions with PqsE and/or RhlR-P37 and to investigate their binding mode. Most compounds interacted with both RhlR-P37 and PqsE. However, no co-crystal structure could be obtained, leaving the precise binding mode unresolved. In summary, this work identified a series of ligands that inhibited the PqsE-RhlR interaction both in vitro and in cellulo, providing a foundation for the development of virulence factor inhibitors that target this interaction and hold potential as novel anti-infectives against P. aeruginosa.