2-Heptyl-4-hydroxyquinoline (HHQ) and Pseudomonas quinolone signal (PQS) are involved in the regulation of virulence factor production and biofilm formation in Pseudomonas aeruginosa. PqsD is a key enzyme in the biosynthesis of these signal molecules. Using a ligand-based approach, we have identified the first class of PqsD inhibitors. Simplification and rigidization led to fragments with high ligand efficiencies. These small molecules repress HHQ and PQS production and biofilm formation in P. aeruginosa. This validates PqsD as a target for the development of anti-infectives.
Pseudomonas aeruginosa employs a characteristic pqs quorum sensing (QS) system that functions via the signal molecules PQS and its precursor HHQ. They control the production of a number of virulence factors and biofilm formation. Recently, we have shown that sulfonamide substituted 2-benzamidobenzoic acids, which are known FabH inhibitors, are also able to inhibit PqsD, the enzyme catalyzing the last and key step in the biosynthesis of HHQ. Here, we describe the further optimization and characterization of this class of compounds as PqsD inhibitors. Structural modifications showed that both the carboxylic acid ortho to the amide and 3'-sulfonamide are essential for binding. Introduction of substituents in the anthranilic part of the molecule resulted in compounds with IC50 values in the low micromolar range. Binding mode investigations by SPR with wild-type and mutated PqsD revealed that this compound class does not bind into the active center of PqsD but in the ACoA channel, preventing the substrate from accessing the active site. This binding mode was further confirmed by docking studies and STD NMR.
Pseudomonas aeruginosa quorum-sensing (QS) is
a sophisticated network of genome-wide regulation triggered in response
to population density. A major component is the self-inducing pseudomonas
quinolone signal (PQS) QS system that regulates the production of
several nonvital virulence- and biofilm-related determinants. Hence,
QS circuitry is an attractive target for antivirulence agents with
lowered resistance development potential and a good model to study
the concept of polypharmacology in autoloop-regulated systems per se. Based on the finding that a combination of PqsR
antagonist and PqsD inhibitor synergistically lowers pyocyanin, we
have developed a dual-inhibitor compound of low molecular weight and
high solubility that targets PQS transcriptional regulator (PqsR)
and PqsD, a key enzyme in the biosynthesis of PQS-QS signal molecules
(HHQ and PQS). In vitro, this compound markedly reduced
virulence factor production and biofilm formation accompanied by a
diminished content of extracellular DNA (eDNA). Additionally, coadministration
with ciprofloxacin increased susceptibility of PA14 to antibiotic
treatment under biofilm conditions. Finally, disruption of pathogenicity
mechanisms was also assessed in vivo, with significantly
increased survival of challenged larvae in a Galleria mellonella infection model. Favorable physicochemical properties and effects
on virulence/biofilm establish a promising starting point for further
optimization. In particular, the ability to address two targets of
the PQS autoinduction cycle at the same time with a single compound
holds great promise in achieving enhanced synergistic cellular effects
while potentially lowering rates of resistance development.
The present work deals with the optimization of an inhibitor of PqsD, an enzyme essential for Pseudomonas aeruginosa quorum sensing apparatus. Molecular docking studies, supported by biophysical methods (surface plasmon resonance, isothermal titration calorimetry, saturation transfer difference NMR), were used to illuminate the binding mode of the 5-aryl-ureidothiophene-2-carboxylic acids. Enabled to make profound predictions, structure-based optimization led to increased inhibitory potency. Finally a covalent inhibitor was obtained. Binding to the active site was confirmed by LC-ESI-MS and MALDI-TOF-MS experiments. Following this rational approach, potent PqsD inhibitors were efficiently developed within a short period of time. This example shows that a combination and careful application of in silico and biophysical methods represents a powerful complement to cocrystallography.
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